Cytodifferentiating agents and histone deacetylase inhibitors, and methods of use thereof

ABSTRACT

The present invention provides the compound having the formula: 
                         
wherein each of R 1  and R 2  is, substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, t-butyl, aryloxy, arylalkyloxy, or pyridine group; wherein A is an amido moiety, —O—, —S—, —NH—, or —CH 2 —; and wherein n is an integer from 3 to 8. The present invention also provides a method of selectively inducing growth arrest, terminal differentiation and/or apoptosis of neoplastic cells and thereby inhibiting proliferation of such cells. Moreover, the present invention provides a method of treating a patient having a tumor characterized by proliferation of neoplastic cells. Lastly, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically acceptable amount of the compound above.

RELATED APPLICATIONS

This application is a divisional of U.S. Application Ser. No.10/281,875, filed on Oct. 25, 2002 now U.S. Pat. No. 7,126,001, which isa continuation of U.S. application Ser. No. 09/645,430, filed Aug. 24,2000, now U.S. Pat. No. 6,511,990, which claims the benefit of U.S.Provisional Application No. 60/208,688, filed Jun. 1, 2000 and U.S.Provisional Application No. 60/152,755, filed Sep. 8, 1999. The contentsof these applications are incorporated herein by reference in theirentireties.

Throughout this application various publications are referenced byArabic numerals within parentheses. Full citation for these publicationsmay be found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Cancer is a disorder in which a population of cells has become, invarying degrees, unresponsive to the control mechanisms which normallygovern proliferation and differentiation. A recent approach to cancertherapy has been to attempt induction of terminal differentiation of theneoplastic cells (1). In cell culture models differentiation has beenreported by exposure of cells to a variety of stimuli, including: cyclicAMP and retinoic acid (2,3), aclarubicin and other anthracylcines (4).

There is abundant evidence that neoplastic transformation does notnecessarily destroy the potential of cancer cells to differentiate(1,5,6). There are many examples of tumor cells which do not respond tothe normal regulators of proliferation and appear to be blocked in theexpression of their differentiation program, and yet can be induced todifferentiate and cease replicating. A variety of agents, including somerelatively simple polar compounds (5,7-9), derivatives of vitamin D andretinoic acid (10-12), steroid hormones (13), growth factors (6, 14),proteases (15, 16), tumor promoters (17,18), and inhibitors of DNA orRNA synthesis (4,19-24), can induce various transformed cell lines andprimary human tumor explants to express more differentiatedcharacteristics.

Early studies by the some of present inventors identified a series ofpolar compounds that were effective inducers of differentiation in anumber of transformed cell lines (8,9). One such effective inducer wasthe hybrid polar/apolar compound N,N′-hexamethylene bisacetamide (HMBA)(9), another was suberoylanilide hydroxamic acid (SAHA) (39, 50). Theuse of these compounds to induce murine erythroleukemia (MEL) cells toundergo erythroid differentiation with suppression of oncogenicity hasproved a useful model to study inducer-mediated differentiation oftransformed cells (5,7-9).

HMBA-induced MEL cell terminal erythroid differentiation is a multistepprocess. Upon addition of HMBA to MEL cells (745A-DS19) in culture,there is a latent period of 10 to 12 hours before commitment to terminaldifferentiation is detected. Commitment is defined as the capacity ofcells to express terminal differentiation despite removal of inducer(25). Upon continued exposure to HMBA there is progressive recruitmentof cells to differentiate. The present inventors have reported that MELcell lines made resistant to relatively low levels of vincristine becomemarkedly more sensitive to the inducing action of HMBA and can beinduced to differentiate with little or no latent period (26).

HMBA is capable of inducing phenotypic changes consistent withdifferentiation in a broad variety of cells lines (5). Thecharacteristics of the drug induced effect have been most extensivelystudied in the murine erythroleukemia cell system (5,25,27,28). MEL cellinduction of differentiation is both time and concentration dependent.The minimum concentration required to demonstrate an effect in vitro inmost strains is 2 to 3 mM; the minimum duration of continuous exposuregenerally required to induce differentiation in a substantial portion(>20%) of the population without continuing drug exposure is about 36hours.

There is evidence that protein kinase C is involved in the pathway ofinducer-mediated differentiation (29). The in vitro studies provided abasis for evaluating the potential of HMBA as a cytodifferentiationagent in the treatment of human cancers (30). Several phase I clinicaltrials with HMBA have been completed (31-36). Clinical trials have shownthat this compound can induce a therapeutic response in patients withcancer (35,36). However, these phase I clinical trials also havedemonstrated that the potential efficacy of HMBA is limited, in part, bydose-related toxicity which prevents achieving optimal blood levels andby the need for intravenous administration of large quantities of theagent, over prolonged periods. Thus, some of the present inventors haveturned to synthesizing compounds that are more potent and possibly lesstoxic than HMBA (37).

Recently, a class of compounds that induce differentiation, have beenshown to inhibit histone deacetylases. Several experimental antitumorcompounds, such as trichostatin A (TSA), trapoxin, suberoylanilidehydroxamic acid (SAHA), and phenylbutyrate have been shown to act, atleast in part, by inhibiting histone deacetylases (38, 39, 42).Additionally, diallyl sulfide and related molecules (43), oxamflatin,(44), MS-27-275, a synthetic benzamide derivative, (45) butyratederivatives (46), FR901228 (47), depudecin (48), and m-carboxycinnamicacid bishydroxamide (39) have been shown to inhibit histonedeacetylases. In vitro, these compounds can inhibit the growth offibroblast cells by causing cell cycle arrest in the G1 and G2 phases(49-52), and can lead to the terminal differentiation and loss oftransforming potential of a variety of transformed cell lines (49-51).In vivo, phenylbutyrate is effective in the treatment of acutepromyelocytic leukemia in conjunction with retinoic acid (53). SAHA iseffective in preventing the formation of mammary tumors in rats, andlung tumors in mice (54, 55).

U.S. Pat. No. 5,369,108 (41) issued to some of the present inventorsdiscloses compounds useful for selectively inducing terminaldifferentiation of neoplastic cells, which compounds have two polar endgroups separated by a flexible chain of methylene groups, wherein one orboth of the polar end groups is a large hydrophobic group. Suchcompounds are stated to be more active than HMBA and HMBA relatedcompounds.

However, U.S. Pat. No. 5,369,108 does not disclose that an additionallarge hydrophobic group at the same end of the molecule as the firsthydrophobic group would further increase differentiation activity about100 fold in an enzymatic assay and about 50 fold in a celldifferentiation assay.

This new class of compounds of the present invention may be useful forselectively inducing terminal differentiation of neoplastic cells andtherefore aid in treatment of tumors in patients.

SUMMARY OF THE INVENTION

The subject invention provides a compound having the formula:

wherein R₁ and R₂ are the same or different and are each a hydrophobicmoiety; wherein R₃ is hydroxamic acid, hydroxylamino, hydroxyl, amino,alkylamino, or alkyloxy group; and n is an integer from 3 to 10, or apharmaceutically acceptable salt thereof.

The subject invention also provides A compound having the formula:

wherein each of R₁ and R₂ is, substituted or unsubstituted, aryl,cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino,9-purine-6-amine, thiazoleamino group, hydroxyl, branched or unbranchedalkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group;wherein R₃ is hydroxamic acid, hydroxylamino, hydroxyl, amino,alkylamino, or alkyloxy group; wherein R₄ is hydrogen, a halogen, aphenyl, or a cycolalkyl moiety; wherein A may be the same or differentand represents an amide moiety, —O—, —S—, —NR₅—, or —CH₂—, where R₅ is asubstituted or unsubstituted C₁-C₅ alkyl; and wherein n is an integerfrom 3 to 10, or a pharmaceutically acceptable salt thereof.

The subject invention also provides a method of selectively inducingterminal differentiation of neoplastic cells and thereby inhibitingproliferation of such cells which comprises contacting the cells undersuitable conditions with an effective amount of the aforementionedcompound.

DESCRIPTION OF THE FIGURES

FIG. 1. The effect of Compound 1 according to the subject invention onMEL cell differentiation.

FIG. 2. The effect of Compound 1 according to the subject invention onHistone Deacetylase 1 activity.

FIG. 3. The effect of Compound 2 according to the subject invention onMEL cell differentiation.

FIG. 4. The effect of Compound 3 according to the subject invention onMEL cell differentiation.

FIG. 5. The effect of Compound 3 according to the subject invention onHistone Deacetylase 1 activity.

FIG. 6. The effect of Compound 4 according to the subject invention onMEL cell differentiation.

FIG. 7. The effect of Compound 4 according to the subject invention onHistone Deacetylase 1 activity.

FIG. 8. A photoaffinity label (3H-498) binds directly to HDAC 1

FIG. 9. SAHA causes accumulation of acetylated histones H3 and H4 in theCWR22 tumor xenograft in mice.

FIG. 10. SAHA causes accumulation of acetylation histones H3 and H4 inperipheral blood monnuclear cells in patients. SAHA was administered byIV infusion daily×3. Samples were isolated before (Pre), followinginfusion (Post) and 2 hours after infusion.

FIGS. 11A-1 through 11A-5; FIGS. 11B-1 through 11B-6; FIGS. 11C-1through 11C-8; FIGS. 11D-1 through 11D-7; FIGS. 11E-1 through 11E-6; andFIGS. 11F-1 through 11F-7. Are graphs showing the effect of selectcompounds on affinity purified human epitope-tagged (Flag) HDAC1.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides a compound having the formula:

wherein R₁ and R₂ are the same or different and are each a hydrophobicmoiety; wherein R₃ is hydroxamic acid, hydroxylamino, hydroxyl, amino,alkylamino, or alkyloxy group; and n is an integer from 3 to 10; or apharmaceutically acceptable salt of the compound.

In the foregoing compound each of R₁ and R₂ is directly attached orthrough a linker, and is, substituted or unsubstituted, aryl,cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino,9-purine-6-amine, thiazoleamino group, hydroxyl, branched or unbranchedalkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group.

Where a linker is used, the linker may be an amide moiety, —O—, —S—,—NH—, or —CH₂—.

According to this invention, n may be 3-10, preferably 3-8, morepreferably 3-7, yet more preferably 4, 5 or 6, and most preferably 5.

In another embodiment of the invention, the compound has the formula:

wherein each of R₁ is, substituted or unsubstituted, aryl, cycloalkyl,cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purine-6-amine,thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl,alkyloxy, aryloxy, arylalkyloxy, or pyridine group. R₂ may be -amide-R₅,wherein R₅ is, substituted or unsubstituted, aryl, cycloalkyl,cycloalkylamino, naptha, pyridineamino, piperdino, 9-purine-6-amine,thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl,alkyloxy, aryloxy, arylalkyloxy, or pyridine group.

In a further embodiment of the invention the compound has the formula:

wherein each of R₁ and R₂ is, substituted or unsubstituted, aryl,cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino,9-purine-6-amine, thiazoleamino group, hydroxyl, branched or unbranchedalkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group;wherein R₃ is hydroxamic acid, hydroxylamino, hydroxyl, amino,alkylamino, or alkyloxy group; wherein R₄ is hydrogen, a halogen, aphenyl, or a cycolalkyl moiety; wherein A may be the same or differentand represents an amide moiety, —O—, —S—, —NR₅—, or —CH₂—, where R₅ is asubstituted or unsubstituted C₁-C₅ alkyl; and wherein n is an integerfrom 3 to 10, or a pharmaceutically acceptable salt thereof.

In another embodiment the compound has the formula:

In yet another embodiment, the compound has the formula:

In a further embodiment, the compound has the formula:

wherein each of R₁ and R₂ is, substituted or unsubstituted, aryl,cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino,t-butyl, aryloxy, arylalkyloxy, or pyridine group; and wherein n is aninteger from 3 to 8.

The aryl or cycloalkyl group may be substituted with a methyl, cyano,nitro, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro,fluoro, bromo, iodo, 2,3-difluoro, 2,4-difluoro, 2,5-difluoro,3,4-difluoro, 3,5-difluoro, 2,6-difluoro, 1,2,3-trifluoro,2,3,6-trifluoro, 2,4,6-trifluoro, 3,4,5-trifluoro, 2,3,5,6-tetrafluoro,2,3,4,5,6-pentafluoro, azido, hexyl, t-butyl, phenyl, carboxyl,hydroxyl, methoxy, phenyloxy, benzyloxy, phenylaminooxy,phenylaminocarbonyl, methyoxycarbonyl, methylaminocarbonyl,dimethylamino, dimethylaminocarbonyl, or hydroxylaminocarbonyl group.

In a further embodiment, the compound has the formula:

or an enantiomer thereof.

In a yet further embodiment, the compound has the formula:

or an enantiomer thereof.

In a further embodiment, the compound has the formula:

or an enantiomer thereof.

In a yet further embodiment, the compound has the formula:

or an enantiomer thereof.

In a further embodiment, the compound has the formula:

or an enantiomer thereof.

In a yet further embodiment, the compound has the formula:

or an enantiomer thereof.

In a yet further embodiment, the compound has the formula:

or an enantiomer thereof.In a further embodiment, the compound has the formula:

or an enantiomer thereof.

In a further embodiment, the compound has the formula:

or an enantiomer thereof.

In a yet further embodiment, the compound has the formula:

or an enantiomer thereof.

In a further embodiment, the compound has the formula:

or an enantiomer thereof.

This invention is also intended to encompass enantiomers and salts ofthe compounds listed above.

In a further embodiment, the compound has the formula:

wherein R₁ and R₂ are the same or different and are each a hydrophobicmoiety:wherein R₅′ is —C(O)—NHOH (hydroxamic acid), —C(O)—CF₃(trifluoroacetyl), —NH—P(O))H—CH₃, —SO₂NH₂ (sulfonamide); —SH (thiol),—C(O)—R₆, wherein R₆ is hydroxyl, amino, alkylamino, or alkyloxy group;and n is an integer from 3 to 10, or a pharmaceutically acceptable saltthereof.

In the foregoing compound, each of R₁ and R₂ may be directly attached orthrough a linker, and is, substituted or unsubstituted, aryl,cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino,9-purine-6-amine, thiazoleamino group, hydroxyl, branched or unbranchedalkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group.

The linker may be an amide moiety, —O—, —S—, —NH—, or —CH₂—.

In another embodiment, the compound has the formula:

wherein each of R₇ is, substituted or unsubstituted, aryl, cycloalkyl,cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purine-6-amine,thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl,alkyloxy, aryloxy, arylalkyloxy, or pyridine group or wherein —NH—R₇ isreplaced by a moiety selected from the group consisting of:

In the foregoing compound, R₂ may be -sulfonamide-R₆, or -amide-R₈,wherein R₈ is, substituted br unsubstituted, aryl, cycloalkyl,cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purine-6-amine,thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl,alkyloxy, aryloxy, arylalkyloxy, or pyridine group.

The R₂ may be —NH—C(O)—Y, —NH—SO₂—Y, wherein Y is selected from thegroup consisting of:

The R₇ may be selected from the group consisting of the following anddesignated R₇′:

In yet another embodiment, the compound has the formula:

wherein R₁ and R₂ are the same or different and are each a hydrophobicmoiety:wherein R₅′ is —C(O)—NHOH (hydroxamic acid), —C(O)—CF₃(trifluoroacetyl), —NH—P(O))H—CH₃, —SO₂NH₂ (sulfonamide), —SH (thiol),—C(O)—R₆,wherein R₆ is hydroxyl, amino, alkylamino, or alkyloxy group; andwherein L is a linker consisting of —(CH₂)—, —C(O)—, —S—, —O—,—(CH═CH)—, -phenyl-, or -cycloalkyl-, or any combination thereof,or a pharmaceutically acceptable salt thereof.

L may also be a linker consisting of —(CH₂)_(n)—, —C(O)—, —S—, —O—,—(CH═CH)_(m)—, -phenyl-, or -cycloalkyl-, or any combination thereof,wherein n is an integer from 3 to 10, and m is an integer from 0 to 10,

In the foregoing compound, n may be from 4-7, and m is from 0-7.Preferably n is 5 or 6, most preferably n is 6. Preferably m is from1-6, more preferably m is 2-5, most preferably m is 3 or 4,

In the compound, each of R₁ and R₂ may be directly attached or through alinker, and is, substituted or unsubstituted, aryl, cycloalkyl,cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purine-6-amine,thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl,alkyloxy, aryloxy, arylalkyloxy, or pyridine group.

The linker may be an amide moiety, —O—, —S—, —NH—, or —CH₂—.

This invention is also intended to encompass enantiomers, salts andpro-drugs of the compounds disclosed herein.

In another embodiment the compound may have the formula:

wherein L is a linker selected from the group consisting of —(CH₂)—,—(CH═CH)—, -phenyl-, -cycloalkyl-, or any combination thereof; andwherein each of R₇ and R₈ are independently substituted orunsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha,pyridineamino, piperidino, 9-purine-6-amine, thiazoleamino group,hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy,arylalkyloxy, or pyridine group.

In a preferred embodiment, the linker L comprises the moiety

In another preferred embodiment, the compound has the formula:

Any of the disclosed compounds can be formed into a pharmaceuticalcomposition together with a pharmaceutically acceptable carrier.

Any of the compounds can also be formed into a pharmaceuticallyacceptable salt of the compound using well known pharmacologicaltechniques.

A prodrug of any of the compounds can also be made using well knownpharmacological techniques.

Any of the compounds can be used in a method of inducing differentiationof tumor cells in a tumor comprising contacting the cells with aneffective amount of the compound so as to thereby differentiate thetumor cells.

Any of the compounds can also be used in a method of inhibiting theactivity of histone deacetylase comprising contacting the histonedeacetylase with an effective amount of the compound so as to therebyinhibit the activity of histone deacetylase.

This invention, in addition to the above listed compounds, is furtherintended to encompass the use of homologs and analogs of such compounds.In this context, homologs are molecules having substantial structuralsimilarities to the above-described compounds and analogs are moleculeshaving substantial biological similarities regardless of structuralsimilarities.

In a further embodiment, the subject invention provides a pharmaceuticalcomposition comprising a pharmaceutically effective amount of any one ofthe aforementioned compounds and a pharmaceutically acceptable carrier.

In a yet further embodiment, the subject invention provides a method ofselectively inducing growth arest, terminal differentiation and/orapoptosis of neoplastic cells and thereby inhibiting proliferation ofsuch cells which comprises contacting the cells under suitableconditions with an effective amount of any one of the aforementionedcompounds.

The contacting should be performed continuously for a prolonged periodof time, i.e. for at least 48 hours, preferably for about 4-5 days orlonger.

The method may be practiced in vivo or in vitro. If the method ispracticed in vitro, contacting may be effected by incubating the cellswith the compound. The concentration of the compound in contact with thecells should be from about 1 nM to about 25 mM, preferably from about 20nM to about 25 mM, more preferably from about 40 nM to 100 μM, yet morepreferably from about 40 nM to about 200 nM. The concentration dependsupon the individual compound and the state of the neoplastic cells.

The method may also comprise initially treating the cells with anantitumor agent so as to render them resistant to an antitumor agent andsubsequently contacting the resulting resistant cells under suitableconditions with an effective amount of any of the compounds above,effective to selectively induce terminal differentiation of such cells.

The present invention also provides a method of treating a patienthaving a tumor characterized by proliferation of neoplastic cells whichcomprises administering to the patient an effective amount of any of thecompounds above, effective to selectively induce growth arrest, terminaldifferentiation and/or apoptosis of such neoplastic cells and therebyinhibit their proliferation.

The method of the present invention is intended for the treatment ofhuman patients with tumors. However, it is also likely that the methodwould be effective in the treatment of tumors in other mammals. The termtumor is intended to include any cancer caused by the proliferation ofneoplastic cells, such as prostate cancer, lung cancer, acute leukemia,multiple myeloma, bladder carcinoma, renal carcinoma, breast carcinoma,colorectal carcinoma, neuroblastoma or melanoma.

Routes of administration for the compound of the present inventioninclude any conventional and physiologically acceptable route, such as,for example, oral, pulmonary, parenteral (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation(via a fine powder formulation or a fine mist), transdermal, nasal,vaginal, rectal, or sublingual routes of administration and can beformulated in dosage forms appropriate for each route of administration.

The present invention also provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, such as sterilepyrogen-free water, and a therapeutically acceptable amount of any ofthe compounds above. Preferably, the effective amount is an amounteffective to selectively induce terminal differentiation of suitableneoplastic cells and less than an amount which causes toxicity in apatient.

The present invention provides the pharmaceutical composition above incombination with an antitumor agent, a hormone, a steroid, or aretinoid.

The antitumor agent may be one of numerous chemotherapy agents such asan alkylating agent, an antimetabolite, a hormonal agent, an antibiotic,colchicine, a vinca alkaloid, L-asparaginase, procarbazine, hydroxyurea,mitotane, nitrosoureas or an imidazole carboxamide. Suitable agents arethose agents which promote depolarization of tubulin. Preferably theantitumor agent is colchicine or a vinca alkaloid; especially preferredare vinblastine and vincristine. In embodiments where the antitumoragent is vincristine, an amount is administered to render the cells areresistant to vincristine at a concentration of about 5 mg/ml. Theadministration of the agent is performed essentially as described abovefor the administration of any of the compounds. Preferably, theadministration of the agent is for a period of at least 3-5 days. Theadministration of any of the compounds above is performed as describedpreviously.

The pharmaceutical composition may be administered daily in 2-6 hourinfusions for a period of 3-21 days, for example, daily in a 4 hourinfusion for a period of 5 days.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS

Examples 1-5 show the synthesis of substituted L-α-aminosuberichydroxamic acids according, to the subject invention, and Examples 6 and7 show the effects of compounds 1-5 on MEL cell differentiation andHistone Deacetylase activity.

Example 1 Synthesis of Compound 1

N-Boc-ω-methyl-(L)-α-aminosuberate, Boc-Asu(OMe) was prepared accordingto a published procedure (40). (“Boc”=t-butoxycarbonyl;“Asu”=α-aminosuberate (or α-aminosuberic acid))

N-Cbz-ω-t-butyl-(L)-α-aminosuberate, dicyclohexylamine salt waspurchased from Research Plus, Bayonne, N.J.

N-Boc-ω-methyl-(L)-α-aminosuberateanilide, Boc-Asu (OMe)—-NHPh

N-Boc-ω-methyl-(L)-α-aminosuberate (493 mg, 1.63 mmoles) was dissolvedunder Ar in 7 mL of dry CH₂Cl₂. EDC (470 mg, 2.45 mmoles) was added,followed by aniline (230 μL, 2.52 mmoles). The solution was stirred atroom temperature for 2 h 30 min, then washed with dilute HCl (pH 2.4,2×5 mL), sat. NaHCO₃ (10 mL), and H₂O (2×10 mL). The product waspurified by column chromatography (Silica gel, Hexanes: AcOEt 3.5:1).The isolated yield was 366 mg (60%).

¹H-NMR and Mass Spectroscopy were consistent with the product.

N-Benzoyl-ω-methyl-(L)-α-aminosuberateanilide, PhCOHN-Asu(OMe)—NHPh

90 mg of N-Bloc-ω-methyl-(L)-α-aminosuberateanilide (0.238 mmoles) weretreated with 3.2 mL of 25% trifluoroacetic acid (TFA) CH₂Cl₂ for 30 min.The solvent was removed and the residue left under high vacuum for 12 h.It was dissolved under Ar in 3 mL of dry CH₂Cl₂ andbenzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate(PyBOP) (149 mg, 0.286 mmoles), benzoic acid (44 mg, 0.357 mmoles) anddiisopropylethylamine (114 μL, 0.655 mmoles). The solution was stirredat room temperature for 1 h. The product was purified by columnchromatography (Silica gel, Hexanes: AcOEt 3:1-2:1) as a white solid: 75mg, 82%.

¹H-NMR and Mass Spectroscopy were consistent with the product.

The foregoing coupling reaction was also successfully accomplished using1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as areagent.

N-Benzoyl-(L)-α-aminosuberoylanilide, PhCONH-Asu(OH)—NHPh

75 mg (0.196 mmoles) of N-benzoyl-aminosuberateanilide were stirred for6 h at 0° C. in 1M NaOH:THF:MeOH 1:1:1. After complete disappearance ofthe starting material, the solution was neutralized (1M HCl) andextracted with AcOEt. The organic phase was collected and dried. Solventremoval yielded the product as a white solid: 67 mg, 93%.

¹H-NMR and Mass Spectroscopy were consistent with the product.

N-Benzoyl-(L)-α-aminosuberoylanilide-ω-hydroxamic acid,PhCONH-Asu(NHOH)—NHPh

To a suspension of 26 mg ofN-benzoyl-ω-methyl-(L)-α-aminosuberateanilide (I2) in 1 mL of dry CH₂Cl₂was added 58 mg of H₂NOTBDPS (H₂NO-t-butyldiphenylsilyl) followed by 22mg of EDC. The reaction was stirred at room temperature for 4 h. Theintermediate protected hydroxamic acid was purified by columnchromatography (silica gel, CH₂Cl₂: MeOH 100:0-98-2). It was deprotectedby treatment with 5% TFA in CH₂Cl₂ for 1 h 30 min. The product wasprecipitated from acetone-pentane.

¹H-NMR (d₆-DMSO, 500 MHz) δ=10.29 (s, 1H), 8.53 (d, 1H), 7.90 (d, 2H),7.60 (d, 2H), 7.53 (m, 1H), 7.46 (t, 2H), 7.28 (t, 2H), 7.03 (t, 2H),4.53 (q, 1H), 1.92 (t, 2H), 1.78 (m, 2H), 1.50-1.25 (m, 6H). ESI-MS: 384(M+1), 406 (M+Na), 422 (M+K)

Example 2 Synthesis of Compound 2N-Nicotinoyl-(L)-α-aminosuberoylanilide-ω-hydroxamic acid,C₅H₄NCO-Asu(NHOH)—NHPh

It was prepared from N-Boc-ω-methyl-L-α-aminosuberate following the sameprocedure used for the benzoyl analog. Yields and chromatographicbehaviour were comparable.

¹H-NMR (d₆-DMSO, 500 MHz) δ=10.30 (s, 1H), 10.10 (s, 1H), 9.05 (m, 1H),8.80 (m, 1H), 8.71 (m, 1H), 8.24 (m, 1H), 7.60 (m, 2H), 7.30 (m, 2H),7.04 (m, 1H), 4.56 (m, 1H), 1.93 (t, 2H), 1.79 (m, 2H), 1.55-1.30 (m,6H). ESI-MS: 385 (M+1), 407 (M+Na).

Example 3 Synthesis of Compound 3N-benzyloxycarbonyl-ω-t-butyl-(L)-aminosuberic acid,N-Cbz-(L)-Asu(OtBu)—OH

N-Cbz-(L)-Asu(OtBu)—OH, dicyclohexylamine salt (100 mg, 0.178 mmol) waspartitioned between 1 M HCl (5 mL) and EtOAc (10 mL). The organic layerwas removed, and the aqueous portion washed with EtOAc (3×3 mL). Theorganic fractions were combined, washed with brine (1×2 mL), and dried(MgSO₄). The mixture was filtered and concentrated to a colorless film(67 mg, 0.176 mmol, 99%). This compound was used immediately in the nextstep.

N-benzyloxycarbonyl-ω-t-butyl-(L)-α-aminosuberateanilide,N-Cbz-(L)-Asu(OtBu)-NHPh

N-Cbz-(L)-Asu(OtBu)—OH (67 mg, 0.176 mmol) was dissolved in dry CH₂Cl₂(2.5 mL). Aniline (17 μL, 0.187 mmol), PyBOP (97 mg, 0.187 mmol), andiPr₂NEt (46 μL, 0.266 mmol) were added and the mixture stirred for 2 h.The reaction was complete as indicated by TLC. The mixture was dilutedwith EtOAc (5 mL) and water (5 mL), and the layers separated. Theaqueous portion was washed with EtOAc (3×3 mL) and the organic fractionscombined. This solution was washed with 1 M HCl (1×2 mL) and brine (1×2mL), dried (MgSO₄), filtered, and concentrated to a crude oil. This waspassed through a plug of silica gel (30% EtOAc/hexanes) to removebaseline impurities, affording the compound (76 mg, 0.167 mmol, 94%).

¹H NMR (CDCl₃, 400 MHz, no TMS) δ 8.20 (br s, 1H), 7.47 (d, 2H), 7.32(m, 5H), 7.28 (t, 2H), 7.08 (t, 1H), 5.39 (d, 1H), 5.10 (m, 2H), 4.26(m, 1H), 2.18 (t, 2H), 1.93 (m, 1H), 1.67 (m, 1H), 1.55 (m, 3H), 1.42(s, 9H), 1.36 (m, 3H).

N-benzyloxycarbonyl-(L)-α-aminosuberateanilide, N-Cbz-(L)-Asu(OH)—NHPh

N-Cbz-(L)-Asu(OtBu)-anilide (76 mg, 0.167 mmol) was dissolved in dryCH₂Cl₂ (5 mL) and TFA (0.5 mL) added dropwise. The reation was completeby TLC after 3 h. The mixture was concentrated in vacuo to give thetitle compound (80 mg, crude). This compound was taken on withoutpurification to the next step.

¹H NMR (DMSO-d₆, 400 MHz) δ 11.93 (br s, 1H), 9.99 (br s, 1H), 7.57 (m,3H), 7.34 (m, 5H), 7.29 (t, 2H), 7.03 (t, 1H), 5.02 (m, 2H), 4.11 (m,1H), 2.17 (t, 2H), 1.61 (m, 2H), 1.46 (m, 2H), 1.27 (m, 4H).

N-benzyloxycarbonyl-(L)-α-aminosuberateanilide ω-hydroxamic acid,N-Cbz-(L)-Asu(NH—OH)—NHPh

N-Cbz-(L)-Asu(OH)-anilide (80 mg, crude) andO-t-butyldiphenylsilyl-hydroxylamine (60 mg, 0.221 mmol) were dissolvedin CH₂Cl₂ (4 mL). To this was added PyBOP (125 mg, 0.241 mmol) andiPr₂NEt (52 μL, 0.302 mmol) and stirred overnight. TLC indicatedreaction completion. The mixture was concentrated in vacuo and thenpassed through a plug of silica gel (50% EtOAc/hexanes) to removebaseline impurities. Evaporation of volatiles afforded 107 mg ofmaterial which was then dissolved in dry CH₂Cl₂ (5 mL) and TFA (0.25 mL)was added. Monitoring by TLC indicated completion after 1.5 h.Concentrated in vacuo to remove all volatiles. The reside was taken upin EtOAc (3 mL), and then hexanes was added slowly to result in theprecipitation of a white gel. The supernatant was removed, and theprecipitate washed with hexanes (3×2 mL). This material was taken todryness under reduced pressure, to afford the title compound (40 mg,0.097 mmol, 59%).

¹H NMR (DMSO-d₆, 400 MHz) δ 10.32 (s, 1H), 10.00 (s, 1H), 8.64 (br s,1H), 7.57 (m, 3H), 7.37 (m, 5H), 7.30 (t, 2H), 7.04 (t, 1H), 5.02 (m,2H), 4.12 (m, 1H), 1.93 (t, 2H), 1.62 (m, 2H), 1.45 (m, 2H), 1.29 (m,4H); ESI-MS 414 (M+1).

Example 4 Synthesis of Compound 4N-benzyloxycarbonyl-(L)-α-aminoxuberoyl-8-quinolinamide-ω-hydroxamicacid

Prepared in similar manner to compound 3.

1H NMR (DMSO-d6, 400 MHz) δ 10.45 (s, 1H), 10.31 (s, 1H), 8.85 (dd, 1H),8.63 (dd, 1H), 8.42 (dd, 1H), 8.13 (dd, 1H), 8.68 (m, 2H), 7.60 (t, 1H),7.37 (m, 2H), 7.28 (m, 2H), 5.10 (m, 2H), 4.24 (m, 1H), 1.93 (t, 2H),1.85 (m, 1H), 1.70 (m, 1H), 1.50 (m, 2H), 1.42 (m, 2H), 1.30 (m, 2H);ESI-MS 465 (M+1).

Example 5 Synthesis of Compound 5N-Benzoyl-(L)-α-aminosuberoyl-8-quinolinamide-ω-hydroxamic acid

A sample of the N-Cbz-ω-t-butyl L-α-aminosuberoyl-8-quinolinamide (90mg, 0.178 mmoles) was obtained from the previous-synthesis. The Cbzgroup was removed by hydrogenation in MeOH on 5% Pd on C. The resultingfree amine was coupled with benzoic acid using EDC in dry CH₂Cl₂ (69%over the two steps). After TFA deprotection of the t-butyl ester, theusual coupling with H₂NOTBDPS followed by deprotection afforded thedesired hydroxamic acid.

¹H-NMR (d₆-DMSO, 500 MHz) δ=10.55 (s, 1H), 10.30 (s, 1H), 9.03 (m, 1H),8.78 (m, 1H), 8.62 (m, 1H), 8.40 (m, 1H0, 7.97 (m, 2H), 7.67-7.46 (m,6H), 4.66 (m, 1H), 1.94 (t, 2H), 1.87 (m, 1H), 1.80-1.20 (m, 7H).ESI-MS: 435 (M+1).

Example 6 Synthesis of Compound with Inverted Amide Group

A compound having the following formula:

is synthesized by treating a malonic ester:

with a base, and then adding:

where X is a halogen, to form:

from which R is removed by reaction with an amine and a carbodiimidereagent ro form:

from which R′ is removed and converted to hydroxamic acid (NHOH) as inthe previous examples.

In the foregoing scheme, R may be t-butyl, removed with trifluoroaceticacid; R′ may be methyl, removed with a base or LiI; and each R″ may bethe same or different, depending on the reagent used.

Example 7 Effect of Compound 1(N-Benzoyl-(L)-α-aminosuberoylanilide-ω-hydroxamic acid,PhCONH-Asu(NHOH)—NHPh) on MEL Cell Differentiation and HistoneDeacetylase Activity

Murine Erythroleukemia (MEL) Cell Differentiation.

The MEL cell differentiation assay was used to assess the ability ofCompound 1 to induce terminal differentiation. MEL cells(logarithmically dividing) were cultured with the indicatedconcentrations of Compound 1. Following a 5-day culture period, cellgrowth was determined using a Coulter Counter and differentiation wasdetermined microscopically using the benzidine assay to determinehemoglobin protein accumulation on a per cell basis.

It was observed, as shown in FIG. 1, that Compound 1 (200 nM) is able toinduce MEL cell differentiation.

Histone Deacetylase (HDAC) Enzymatic Activity.

The effect of Compound 1 on affinity purified human epitope-tagged(Flag) HDAC1 was assayed by incubating the enzyme preparation in theabsence of substrate on ice for 20 min with the indicated amounts ofCompound 1. Substrate ([³H]acetyl-labeled murine erythroleukemiacell-derived histone) was added and the samples were incubated for 20min at 37° C. in a total volume of 30 μl. The reactions were thenstopped and released acetate was extracted and the amount ofradioactivity released determined by scintillation counting.

It was observed, as shown in FIG. 2, that Compound 1 is a potentinhibitor of HDAC1 enzymatic activity (ID₅₀=1 nM).

Example 8 Effect of Compound 2(N-Nicotinoyl-(L)-α-aminosuberoylanilide-ω-hydroxamic acid,C₅H₄NCO-Asu(NHOH)—NHPh) on MEL Cell Differentiation

Murine Erythroleukemia (MEL) Cell Differentiation:

The MEL cell differentiation assay was used to assess the ability ofCompound 2 to induce terminal differentiation. MEL cells(logarithmically dividing) were cultured with the indicatedconcentrations of Compound 2. Following a 5-day culture perioddifferentiation was determined microscopically using the benzidine assayto determine hemoglobin protein accumulation on a per cell basis.

It was observed, as shown in FIG. 3, that Compound 2 (800 nM) is able toinduce MEL cell differentiation.

Example 9 Effect of Compound 3(N-benzyloxycarbonyl-(L)-α-aminosuberateanilide ω-hydroxamic acid,N-Cbz-(L)-Asu(NH—OH)—NHPh) on MEL Cell Differentiation and HistoneDeacetylase Activity

Murine Erythroleukemia (MEL) Cell Differentiation:

The MEL cell differentiation assay was used to assess the ability ofCompound 3 to induce terminal differentiation. MEL cells(logarithmically dividing) were cultured with the indicatedconcentrations of Compound 3. Following a 5-day culture perioddifferentiation was determined microscopically using the benzidine assayto determine hemoglobin protein accumulation on a per cell basis.

It was observed, as shown in FIG. 4, that Compound 3 (400 nM) is able toinduce MEL cell differentiation.

Histone Deacetylase (HDAC) Enzymatic Activity:

The effect of Compound 3 on affinity purified human epitopetagged (Flag)HDAC1 was assayed by incubating the enzyme preparation in the absence ofsubstrate on ice for 20 min with the indicated amounts of HPC. Substrate([³H]accetyl-labelled murine erythroleukemia cell-derived histone) wasadded and the samples were incubated for 20 min at 37° C. in a totalvolume of 30 μl. The reactions were then stopped and released acetatewas extracted and the amount of radioactivity released determined byscintillation counting.

It was observed, as shown in FIG. 5, that Compound 3 is a potentinhibitor of HDAC1 enzymatic activity (ID₅₀-100 nM)

Example 10 Effect of Compound 4(N-benzyloxycarbonyl-(L)-α-aminoxuberoyl-8-quinolinamide-ω-hydroxamicacid) on MEL Cell Differentiation and Histone Deacetylase Activity

Murine Erythroleukemia (MEL) Cell Differentiation:

The MEL cell differentiation assay was used to assess the ability ofCompound 4 to induce terminal differentiation. MEL cells(logarithmically dividing) were cultured with the indicatedconcentrations of Compound 4. Following a 5-day culture perioddifferentiation was determined microscopically using the benzidine assayto determine hemoglobin protein accumulation on a per cell basis.

It was observed, as shown in FIG. 6, that Compound 4 (40 nM) is able toinduce MEL cell differentiation.

Histone Deacetylase (HDAC) Enzymatic Activity:

The effect of Compound 4 on affinity purified human epitope-tagged(Flag) HDAC1 was assayed by incubating the enzyme preparation in theabsence of substrate on ice for 20 min with indicated amounts of HPC.Substrate ([³H]acetyl-labelled murine erythroleukemia cell-derivedhistone) was added and the samples were incubated for 20 min at 37° C.in a total volume of 30 μl. The reactions were then stopped and releasedacetate was extracted and the amount of radioactivity releaseddetermined by scintillation counting.

It was observed, as shown in FIG. 7, that Compound 4 is a potentinhibitor of HDAC1 enzymatic activity (ID₅₀<10 nM).

SAHA inhibits the activity of affinity purified HDAC1 and HDAC3 (39).Crystallographic studies with SAHA and a HDAC related protein revealthat SAHA inhibits HDAC by a direct interaction with the catalytic site(66). Additional studies demonstrate that a tritium labeledphotoaffinity SAHA analog (³H-498) that contains an azide moiety (67)binds directly to HDAC1 (FIG. 8). These results indicate that this classof hydroxamic acid based compound inhibits HDAC activity through adirect interaction with the HDAC protein.

SAHA causes the accumulation of acetylated histones H3 and H4 in vivo.The in vivo effect of SAHA has been studied using the CWR22 humanprostate xenograft in mice (68). SAHA (50 mg/kg/day) caused a 97%reduction in mean final tumor volume compared to controls with noapparent toxicity. SAHA administration at this dose caused an increasein acetylated histones H3 and H4 in the tumor xenograft (FIG. 9).

SAHA is currently in Phase I Clinical Trials in patients with solidtumors. SAHA causes an accumulation of acetylated histones H3 and H4 inthe peripheral blood mononuclear cells isolated from patients undergoingtreatment (FIG. 10).

Table 1 shows a summary of the results of the Examples 7-10, testingcompounds 1-4, and also compares the results to the results obtainedfrom using SAHA.

TABLE 1 Summary of Test results of compounds 1–4, and comparison to SAHAresults. MEL HDAC Com- Differentiation Inhibition pound Range Opt. % B+Range ID50 1 0.1 to 5.0 μM 200 nM 44% 0.0001 to 100 μM  1 nM 2 0.2 to12.5 μM 800 nM 27% TBT 3 0.1 to 50 μM 400 nM 16% 0.01 to 100 μM 100 nM 40.01 to 50 μM  40 nM  8% 0.01 to 100 μM <10 nM SAHA 2500 nM  68% 0.01 to100 μM 200 nM

Example 12 Modified Inhibitors of HDAC

In additional studies we found that compounds 6 and 7 shown below werevery effective inhibitors of the enzyme HDAC. Compound 6 had ID₅₀ of 2.5nM, and compound 7 had ID₅₀ of 50 nM. This contrasts with an ID₅₀ forSAHA of 1 μM, much higher. Note that the 1 μM ID₅₀ for SAHA as aninhibitor of HDAC is of the same general magnitude as its 2.5 μM optimaldose for the cytodifferentiation of MEL cells, but this close similarityis not true for all the compounds examined. In some cases very effectiveHDAC inhibitors are less effective as cytodifferentiaters, probablybecause the drugs are metabolized in the cell assays. Also, all celltypes are not the same, and some compounds are much better against humantumor cells such as HT-29 than they are against MEL cells. Thus,inhibition of HDAC cells is a preliminary indicator.

Example 13 Evolution of Compounds Without a Hydroxamic Acid Portion

Of the above compounds which are hydroxamic acids, we have found thatthey undergo enzymatic hydrolysis rather rapidly to the carboxylicacids, so their biological lifetimes are short. We were interested inevolving compounds which might be more stable in vivo. Thus we havedeveloped inhibitors of HDAC that are not hydroxamic acids, and that canbe used as cytodifferentiating agents with longer biological lifetimes.Furthermore, we found that the newly evolved compounds have betterselectivity to HDAC than, e.g. SAHA.

We have evolved compounds that have double bonds, similarly toTrichostatin A (TSA) to see if the resulting compounds have even greaterefficacy. Also, the chain in TSA is only five carbons, not the six ofSAHA. In Oxamflatin there is a chain of four carbons containing a doublebond and an ethinyl link between the hydroxamic acid and the firstphenyl ring, and Oxamflatin has been claimed to be an effectiveinhibitor of HDAC. We incorporate some of these features in ourcompounds, including those compounds that are not hydroxamic acids.

Also disclosed are simple combinatorial methods for screening a varietyof such compounds for efficacy and selectivity with respect to HDACinhibition.

Furthermore, since there are many important enzymes that contain Zn(II),hydroxamic acids, and perhaps some of the other metal coordinatinggroups, can also bind to Zn(II) and other metals.

Since the target for HDAC is an acetyllysine sidechain of histone, wemake compounds in which transition state analogs of the substrate arepresent. For example, we synthesize compounds like SAHA in which thehydroxamic acid group —CO—NHOH is replaced by a trifluoroacetyl group,—CO—CF₃. The resulting 8 will easily form a hydrate, and thus bind tothe Zn(II) of HDAC in a mimic 9 of the transition state 10 fordeacetylation. This is related to the work published by Lipscomb [56] onthe binding to carboxypeptidase A of a substrate analog 11 containing aCF₃—CO—CH₂ group in place of the normal amide. The hydrate of the ketonecoordinated to the Zn(II) as a mimic of the transition state forcatalyzed hydrolysis of an amide substrate. Our synthesis of aparticular example 12 in the fluoroketone series is shown in Schemebelow:

After the malonic ester alkylation, the aldehyde is prepared and thenconverted to the trifluoromethyl carbinol with Rupperts reagent [57,58]. The malonic bis-anilides are prepared, and the carbinol oxidized tothe ketone 12 with the Dess-Martin reagent [59]. Other approaches weretried unsuccessfully. In particular, attempts to convert a carboxylicacid derivative directly to a trifluoromethyl ketone did not work.

Compound 12 has been tested with HDAC and found to be an inhibitor ofthe enzyme. Thus, we also adapt this synthesis to the preparation ofanalogs of 12 with unsaturation, etc., in the chain, and other groups atthe left end of the molecule.

Example 14 Evolution of Compounds Where the Hydroxamic Acid Group isReplaced by NH—P(O)OH—CH₃

An analog of SAHA in which the CH₂—CO—NHOH group is replaced byNH—P(O)OH—CH₃ may be synthesized by the general scheme shown below. Theresulting compound, 13, binds to the Zn(II) of HDAC the way a relatedgroup binds to the Zn(II) of carboxypeptidase in analogs such as thatprepared by Bartlett [60].

A classic inhibitor of the Zn(II) enzyme carbonic anhydrase is asulfonamide, whose anion binds to the Zn(II) [61]. Thus compound 14, ananalog of SAHA with a sulfonamide group, is synthesized as shown below.In the last step we react a carboxylic sulfonic bis-chloride withaniline and ammonia. Since the carboxylic acid chloride reacts faster,we use the sequence of aniline, then ammonia, but the sequence may bereversed, or the mixture may be separated if the two are of similarreactivity.

In the course of the synthesis of 14, we use a thiol 15 easily made fromthe corresponding haloacid. Thiols are also inhibitors of Zn(II) enzymessuch as carboxypeptidase A and related peptidases such as AngiotensinConverting Enzyme (ACE), so we convert 15 to 16 as an inhibitor of HDAC.A similar synthesis can be used to attach the NH—P(O)OH—CH₃ group toother compounds, in particular compounds 6 and 7.

Example 15 Varying the Linker Between the Zn(II) Binding Group and theHydrophobic Binding Groups

Based on the results with Oxamflatin, it seems that a phenyl ring can bepart of the chain between the Zn(II) binding group and the left handsection of the molecule as drawn, particularly when the phenyl ring ismeta substituted. Thus, we provide a synthesis to incorporate such metasubstituted chains into other of our compounds. We construct compounds17 and 18. The simple syntheses, not shown in detail, only require thatinstead of the hydroxamic acid attached to the phenyl ring we make thearyl amides of 17 and 18.

Additional compounds may be synthesized, such as 19 and 20 toincorporate the trifluoromethyl ketone group of 12 that we know iseffective as a Zn(II) binder in HDAC. The syntheses involve preparingcompounds 21 and 22 and then adding CF₃ to form the carbinol, followedby oxidation as in the synthesis of 12. A simple synthesis involves Heckcoupling of compounds 23 and 24 with ethyl acrylate, and conversion ofthe ester to aldehydes 21 and 22 by reduction to the carbinol and thenreoxidation.

All the chains shown so far contain only carbon atoms, but thioetherlinks may be acceptable and even useful, and they add synthetic ease.Thus, sulfonamides such as 25 and 26, related to 19 and 20, from thecorresponding thiophenol and bromomethylsulfonamide. A related synthesismay be used to make the corresponding phosphonamidates 27 and 28, ifthis class proves to be useful HDAC inhibitors and cytodifferentiators.In this case, (N-protected) m-aminobenzoic acid is used to acylate thearylamines, then phosphorylate the anilino group.

Example 16 Varying the Left Hand of the Molecule, Carrying theHydrophobic Groups

To vary the hydrophobic groups, we synthesized compound 29, as anintermediate that can be treated with various amines to make thecompounds 30. Then deprotection of the hydroxamic acid group willgenerate the general class 31. The synthesis is shown in the schemebelow.

In the synthesis the O-protected hydroxylamine is acylated withbromohexanoic acid, and the compound then alkylates the bis-pentafluoroester of malonic acid. The resulting 29 then reacts with various amines,and the protecting group is removed with acid.

With this compound as the starting material, we synthesize relatedlibraries carrying the other Zn(II) binding groups. For example,alkylation of the malonate with compound 32 lets us make aphosphonamidate library, and compound 33 will let us make a CF₃—COlibrary. In a similar way, a sulfonamide library can be made if the workdescribed earlier indicates that this is a promising Zn(II) bindinggroup for HDAC. Of course after malonate alkylation and aminolysis thecompound from 32 will be demethylated, while that from 33 will beoxidized.

This also allows to expand on the structure of compound 6, thederivative of aminosuberic acid. As described, this was one of the mosteffective HDAC inhibitor we have examined. We prepared this compoundusing an enzymatic hydrolysis to achieve optical resolution andselectivity among the two carbomethoxy groups of 34, so that we couldconvert one of them to the aminoquinoline amide of 6 while protectingthe nitrogen as a carbobenzoxy group. At the end of the synthesis weconverted the remote carbomethoxy group to a hydroxamate. However, 6 isan intermediate that can be used to prepare other derivatives. Thecarbobenzoxy group from 6 can be removed and the amine 35 can beacetylated with a variety of carboxylic acids to prepare library 36, orsulfonic acid chlorides to prepare the corresponding sulfonamides.

Also, we synthesize a different library of amides 37 related to 6, andthen expand it with a library of other amides 38 by acylating the aminogroup after deprotection. We also synthesize a group of compounds 39 inwhich after the carbobenzoxy group of 37 is removed we make a library ofsulfonamides using various sulfonyl chlorides. In all this, it thehydroxamic acid group may be protected.

The foregoing synthesis schemes can be used to generate compounds havinga large number of variation. Some substituent groups that are likely toresult in compounds having potential good affinity to HDAC or having gotdifferentiating activity are as follows:

Some Amines that can be Incorporated in Place of the Aniline in SAHA, oras the X Group in Compounds 37 and 38:

Some Carboxylic and Sulfonic Acids that can be Incorporated as GroupY—CO in Compound 38 or 39:

Example 17 Synthesis Using the Foregoing Schemes

Reagents and starting materials were obtained from commercial suppliersand used without further purification unless otherwise indicated. Formoisture-sensitive reactions, solvents were freshly distilled prior touse: tetrahydrofuran was distilled under argon from sodium metalutilizing benzophenone as an indicator; dichloromethane and acetonitrilewere distilled from powdered calcium hydride. Anhydrous benzene,anhydrous DIEA, and anhydrous pyridine were drawn by syringe from asealed bottle purchased from Aldrich. tert-Butanol was dried over 4 Åmolecular sieves before use. Sodium hydride was purchased as a 60%dispersion in mineral oil. Aniline, diisopropylamine, N-methylaniline,and benzyl alcohol were freshly distilled before use. Deuteratedsolvents were obtained from Cambridge Isotope Laboratories. Air- and/ormoisture-sensitive reactions were carried out under an atmosphere of dryargon in oven- or flame-dried glassware equipped with a tightly-fittingrubber septum. Syringes and needles were oven-dried before use.Reactions at 0° C. were carried out in an ice/water bath. Reactions at−78° C. were carried out in a dry ice/acetone bath.

Chromatography

Analytical thin-layer chromatography (TLC) was conducted on glass platesprecoated with silica gel 60 F-254, 0.25 mm thickness, manufactured byEM Science, Germany. Eluted compounds were visualized by one or more ofthe following: short-wave ultraviolet light, I₂ vapor, KMnO₄ stain, orFeCl₃ stain. Preparative TLC was carried out on Whatman precoated platesof either 500 μm or 1000 μm silica gel thickness. Flash columnchromatography was performed on Merck Kieselgel 60, 230-400 mesh.

Instrumentation

NMR spectra were measured on Bruker DPX300 and DRX400 spectrometers; ¹Hwas observed at 300 and 400 MHz, and ¹⁹F at 376 MHz. Chemical shifts arereported as δ values in ppm relative to the solvent residual peak. Massspectra were obtained on a Nermag R-10-1 instrument for chemicalionization (CI) or electron impact ionization (EI) spectra, and on aJeol JMS LCmate for electrospray ionization (ESI+) spectra. CI spectrawere run with either ammonia (NH₃) or methane (CH₄) as the ionizationgas.

(E,E)-7-t-Butoxycarbonyl-octa-2,4-dienedioic acid 8-t-butyl ester1-methyl ester (40)

To a stirred solution of NaH (60% disp., 234 mg, 5.85 mmol) in THF (35mL) at 0° C. was added di-t-butyl malonate (1.20 mL, 5.37 mmol)dropwise. Gas evolution was observed, and the solution was allowed towarm to ambient temperature and stirred for 6 h. A solution of methyl6-bromo-2,4-hexadienoate (62) (1.00 g, 4.88 mmol) in THF (20 mL) wasprepared in a separate flask and stirred in a water bath. To this wascannulated dropwise the malonate mixture, and the reaction allowed toproceed overnight. The reaction was quenched with sat. NH₄Cl (5 mL),then H₂O (10 mL) was added and the mixture extracted with Et₂O (3×15mL). The organic fractions were combined and washed with H₂O (1×10 mL),then with brine, dried over MgSO₄, and filtered. Evaporation underreduced pressure followed by flash chromatography (0-20% EtOAc/hexanes)gave 40 as a clear colorless oil (850 mg, 2.49 mmol, 51%). TLC R_(f)0.66 (20% EtOAc/hexanes); ¹H-NMR (CDCl₃, 400 MHz) δ 7.26 (dd, 1H), 6.26(dd, 1H), 6.10 (m, 1H), 5.82 (d, 1H), 3.78 (s, 3H), 3.12 (t, 1H), 2.64(t, 2H), 1.41 (s, 18H).

(E,E)-7-Carboxy-octa-2,4-dienedioic acid 1-methyl ester (41)

To a stirred solution of 40 (200 mg, 0.59 mmol) in CH₂Cl₂ (10 mL) wasadded TFA (1 mL). The reaction was allowed to proceed overnight.Volatiles were removed under reduced pressure to leave 41 as a whitesolid (112 mg, 0.49 mmol, 83%). ¹H-NMR (CD₃OD, 400 MHz) δ 7.11 (dd, 1H),6.33 (dd, 1H), 6.16 (m, 1H), 5.81 (d, 1H), 3.76 (s, 3H), 3.15 (t, 1H),2.70 (t, 2H).

4-Pentenoic acid phenylamide (42)

To a stirred solution of oxalyl chloride (2.0 M in CH₂Cl₂, 11.5 mL, 23.1mmol) in CH₂Cl₂ (100 mL) and DMF (1 drop) at 0° C. was added 4-pentenoicacid (2.25 mL, 22.0 mmol). This was allowed to warm to ambienttemperature. Upon cessation of gas evolution, the mixture was returnedto 0° C. and a solution of aniline (2.00 mL, 22.0 mmol) and TEA (6.72mL, 26.3 mmol) in CH₂Cl₂ (5 mL) was added dropwise. After warming toambient temperature, the reaction was allowed to proceed for 3 h. Themixture was concentrated under reduced pressure, and then partitionedbetween HCl (1 N, 10 mL) and EtOAc (30 mL) and the layers separated. Theaqueous portion was extracted with EtOAc (3×15 mL) and the organiclayers combined, washed with brine, dried over MgSO₄, and filtered.Concentration under reduced pressure gave a yellowish solid, which wasrecrystallized with toluene to obtain 42 as white crystals (1.97 g,11.24 mmol, 51%). TLC R_(f) 0.68 (50% EtOAc/hexanes); ¹H-NMR (300 MHz,CDCl₃) δ 7.49 (d, 2H), 7.29 (t, 2H), 7.08 (t, 1H), 5.88 (m, 1H), 5.10(dd, 2H), 4.42 (br s, 4H).

(E,E)-Octa-2,4-dienedioic acid 8-t-butyl ester 1-methyl ester (43)

To a stirred solution of diisopropylamine (2.06 mL, 14.7 mmol) in THF(25 mL) at −78° C. was added n-BuLi (2.0 M in hexanes, 6.2 mL, 12.4mmol) and allowed to stir 20 min at this temperature. A solution ofphosphonate 43a (63) (2.66 g, 11.3 mmol) in THF (4 mL) was then addeddropwise, giving a deep yellow color upon addition. After 20 min at −78°C., the mixture was warmed to 0° C. and a solution of aldehyde 43b (64)(1.78 g, 11.3 mmol) in THF (4 mL) was added dropwise. After addition thesolution was allowed to warm to ambient temperature and stirredovernight. It was diluted with Et₂O (30 mL) and washed with H₂O (3×10mL). The aqueous washings were combined and extracted with Et₂O (2×10mL), and the organic portions combined, washed with brine, dried overMgSO₄, and filtered. Evaporation under reduced pressure followed byflash chromatography (10-20% EtOAc/hexanes) gave 43 as a clear oil (1.54g, 57%). TLC R_(f) 0.56 (20% EtOAc/hexanes); ¹H-NMR (400 MHz, CDCl₃) δ7.22 (dd, 1H), 6.19 (dd, 1H), 6.08 (m, 1H), 5.77 (d, 1H), 2.42 (m, 2H),2.32 (t, 2H), 1.42 (s, 9H).

(E,E)-7-Phenylcarbamoyl-hepta-2,4-dienoic acid methyl ester (44)

To a stirred solution of diester 43 (1.00 g, 4.61 mmol) in CH₂Cl₂ (40mL) was added TFA (4.0 mL) and let react for 6 h. The mixture wasconcentrated under reduced pressure to remove volatiles. A white solidconsisting of the crude acid (710 mg, 3.85 mmol) remained. This acid(400 mg, 2.17 mmol) was dissolved in CH₂Cl₂ (20 mL) and to this stirredsolution were added DMAP (13 mg), aniline (218 μL, 2.39 mmol), and EDC(500 mg, 2.61 mmol). After 1.5 h; the mixture was diluted with EtOAc andwashed with H₂O. The layers were separated, and the aqueous extractedwith EtOAc (3×15 mL). The organic portions were combined and washed withHCl (1 N, 1×5 mL) and brine, dried over MgSO₄, and filtered.Concentration under reduced pressure left a brown solid. This wasdissolved in a minimum of CH₂Cl₂, then passed through a plug of silicagel (20-30% EtOAc/hexanes, 200 mL) to remove baseline impurities. Theeluent was concentrated to a light brown oil which was taken up in asmall amount of CH₂Cl₂ and from which crystals were precipitated uponthe addition of hexanes/diethyl ether. The mother liquor was drawn off,the crystals rinsed with ether, and the liquid fraction concentrated andthis procedure repeated several times to ultimately give 44 as off-whitecrystals (324 mg, 1.25 mmol, 58%). TLC R_(f) 0.44 (50% EtOAc/hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.47 (d, 1H), 7.30 (t, 2H), 7.24 (m, 1H), 7.09(t, 1H), 6.24 (dd, 1H), 6.14 (m, 1H), 5.81 (d, 1H), 3.72 (s, 3H), 2.60(m, 2H), 2.47 (t, 2H).

(E,E)-7-(Methyl-phenyl-carbamoyl)-hepta-2,4-dienoic acid methyl ester(45)

The crude acid intermediate from the first step of the preparation of 44(200 mg, 1.09 mmol) and N-methylaniline (130 μL, 1.19 mmol) weredissolved in CH₂Cl₂ (10 mL) and stirred. EDC (271 mg, 1.41 mmol) andDMAP (5 mg) were then added and the reaction run overnight. The mixturewas partitioned between H₂O and EtOAc and the layers separated. Theaqueous layer was extracted with EtOAc (3×10 mL), the organic portionscombined and washed with HCl (1 N, 1×5 mL), then brine, dried overMgSO₄, and filtered. Evaporation under reduced pressure left pure 45 asa brown oil (286 mg, 1.05 mmol, 96%). TLC R_(f) 0.81 (5% MeOH/CH₂Cl₂);¹H-NMR (300 MHz, CDCl₃) δ 7.40 (t, 2H), 7.35 (t, 1H), 7.20 (d, 2H), 7.15(dd, 1H), 6.20 (m, 2H), 5.76 (d, 1H), 3.70 (s, 3H), 3.24 (s, 3H), 2.42(m, 2H), 2.18 (t, 2H).

(E,E)-7-Phenylcarbamoyl-hepta-2,4-dienoic acid (46)

Ester 45 (260 mg, 0.95 mmol) was dissolved in MeOH (7.5 mL). A solutionof LiOH.H₂O (200 mg, 4.76 mmol) in H₂O (2.5 mL) was then added and themixture stirred for 6 h. The reaction was acidified with HCl (1 N) untilpH 2 and then extracted with EtOAc (3×10 mL). The organic fractions werecombined and washed with H₂O and brine, dried over MgSO₄, and filtered.Evaporation under reduced pressure left the product pure 46 as a brownsolid (200 mg, 0.77 mmol, 81%). TLC R_(f) 0.13 (40% EtOAC/hexanes);¹H-NMR (300 MHz, CD₃OD) δ 7.47 (t, 2H), 7.41 (d, 1H), 7.28 (d, 2H), 7.19(dd, 1H), 6.18 (dd, 1H), 6.05 (m, 1H), 3.27 (s, 3H), 3.40 (m, 2H), 2.22(t, 2H).

(E,E)-Octa-2,4-dienedioic acid 1-hydroxyamide 8-phenylamide (47)

Acid 46 (200 mg, 0.77 mmol) and TBDPSO-NH₂ (220 mg, 0.81 mmol) weredissolved in CH₂Cl₂ (8 mL). To this stirred solution were added EDC (178mg, 0.93 mmol) and DMAP (5 mg) and the reaction allowed to proceedovernight. The mixture was concentrated and then passed through a plugof silica gel (EtOAc). Evaporation under reduced pressure left a lightbrown oil (383 mg, 0.75 mmol, 97%). The protected hydroxamate (270 mg,0.53 mmol) was, dissolved in CH₂Cl₂ (10 mL) and TFA was added (0.5 mL).The solution was stirred for 2 h, and a new spot on TLC was observedwhich stained with FeCl₃. The solution was concentrated under reducedpressure and diethyl ether added, giving a residue which adhered to theflask. The liquid phase was drawn off, the residue was triturated withEtOAc, the liquid removed, and evaporation of all volatiles from theresidue gave 47 as a brown gum (23 mg, 0.084 mmol, 16%). TLC R_(f) 0.22(5% MeOH/CH₂Cl₂); ¹H-NMR (400 MHz, CD₃OD) δ 7.50 (t, 2H), 7.40 (t, 1H),2.27 (d, 2H), 7.08 (m, 1H), 6.11 (m, 1H), 5.97 (m, 1H), 5.80 (m, 1H),3.23 (s, 3H), 3.39 (m, 2H), 2.21 (t, 2H).

Octanedioic acid hydroxyamide phenylamide (48)

The title compound 48 was obtained as a brown gum (9 mg) by a series ofsteps analogous to the preparation of 47. TLC R_(f) 0.20 (5%MeOH/CH₂Cl₂); ¹H-NMR (400 MHz, CD₃OD) δ 7.51 (t, 2H), 7.41 (t, 1H), 7.30(d, 2H), 3.29 (s, 3H), 2.11 (m, 4H), 1.58 (m, 4H), 1.22 (m, 4H).

Octanedioic acid benzylamide (49)

To a stirred solution of suberoyl chloride (1.00 mL, 5.55 mmol) in THF(40 mL) at 0° C. was added a solution of benzylamine (0.61 mL, 5.55mmol) and DIEA (1.45 mL, 8.33 mmol) in THF (10 mL) dropwise. The mixturewas allowed to warm to ambient temperature and stirred for 1 h. Then,HCl (10 mL, 1 N) was added and the mixture stirred for 0.5 h. Thecontents were diluted with EtOAc (30 mL) and the layers separated. Theaqueous portion was extracted with EtOAc (3×10 mL), the organicscombined, washed with brine (5 mL), and dried over MgSO₄. Filtration andconcentration under reduced pressure left 49 as an off-white solid.¹H-NMR (300 MHz, DMSO-d₆) δ 11.98 (br s, 1H), 9.80 (t, 1H), 7.32 (m,2H), 7.23 (m, 3H), 4.25 (d, 2H), 2.19 (t, 2H), 2.12 (t, 2H), 1.50 (m,4H), 1.25 (m, 4H).

Octanedioic acid benzylamide hydroxyamide (50)

This compound was prepared from 49 through its protected hydroxamate asdescribed for earlier compounds. Obtained 50 as a white solid. ¹H-NMR(400 MHz, DMSO-d₆) δ 10.30 (s, 1H), 8.27 (t, 1H), 7.28 (m, 2H), 7.23 (m,3H), 5.65 (d, 2H), 2.11 (t, 2H), 1.91 (t, 2H), 1.46 (m, 4H), 1.23 (m,4H).

(7S)-7-Benzyloxycarbonylamino-7-phenylcarbamoyl-heptanoic acid t-butylester (51)

N-Cbz-L-2-aminosuberic acid 8-t-butyl ester, dicyclohexylamine salt (100mg, 0.18 mmol) was dissolved in HCl (5 mL, 1 N) and extracted with EtOAc(3×10 mL). The extracts were combined, washed with brine, and dried overMgSO₄. Evaporation left the free acid as a white solid (68 mg, 0.179mmol). This was dissolved in CH₂Cl₂ (2.5 mL), to which were addedaniline (17 μL, 0.19 mmol), DIEA (46 μL, 0.27 mmol), and finally Py.BOP(97 mg, 0.19 mmol). The solution was stirred for 1 h, then concentrated,and the residue partitioned between H₂O (5 mL) and EtOAc (10 mL). Thelayers were separated, and the aqueous portion extracted with EtOAc(3×10 mL). The extracts were pooled and washed with HCl (1 N), thenbrine, dried over MgSO₄, and filtered. Concentration under reducedpressure gave a solid residue which was passed through a plug of silicagel (30% EtOAc/hexanes). The collected eluent was evaporated to give 51as a white solid (76 mg, 0.167 mmol, 94%). TLC R_(f) 0.38 (30%EtOAc/hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.48 (d, 2H),7.32 (m, 5H), 7.28 (t, 2H), 7.08 (t, 1H), 5.39 (br d, 1H), 5.10 (m, 2H),4.26 (br dd, 1H), 2.07 (t, 2H), 1.92 (m, 1H), 1.66 (m, 1H), 1.55 (m,2H), 1.42 (s, 9H), 1.38 (m, 4H).

(7S)-7-Benzyloxycarbonylamino-7-phenylcarbamoyl-heptanoic acid (52)

To a solution of ester 51 (76 mg, 0.167 mmol) in CH₂Cl₂ (5 mL) was addedTFA (0.5 mL) and the reaction solution stirred for 5 h. The solution wasconcentrated under reduced pressure to give crude 52 as a white solid(80 mg) which was used in the next step without purification. TLC R_(f)0.32 (5% MeOH/CH₂Cl₂); ¹H-NMR (400 MHz, DMSO-d₆) δ 11.93 (br s, 1H),9.99 (s, 1H), 7.58 (d, 2H), 7.55 (d, 1H), 7.35 (m, 4H), 7.29 (t, 2H),7.03 (t, 1H), 5.02 (m, 2H), 4.11 (br dd, 1H), 2.17 (t, 2H), 1.59 (m,2H), 1.48 (m, 2H), 1.22 (m, 4H).

(1S)-(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acid benzylester (53)

To a solution of crude acid 52 (80 mg) and TBDPSO-NH₂ (60 mg, 0.221mmol) in CH₂Cl₂ were added DIEA (52 μL, 0.302 mmol) followed by Py.BOP(125 mg, 0.241 mmol). The solution was stirred for 3 h, thenconcentrated under reduced pressure. The residue was passed through aplug of silica gel (50% EtOAc/hexanes) and the collected eluentevaporated. A white foam (107 mg, 0.164 mmol, 82%) was obtained, thiswas dissolved in CH₂Cl₂ (5 mL) and TFA (0.25 mL) was added and thesolution stirred for 2 h. A new spot that stained with FeCl₃ wasindicated by TLC analysis. The mixture was concentrated under reducedpressure, and the residue was solvated in a minimum of EtOAc and theproduct precipitated with hexanes. The resulting white gel was rinsedwith hexanes and dried under vacuum, to give 53 as a white solid (40 mg,0.097 mmol, 58% over three steps). ¹H-NMR (400 MHz, DMSO-d₆) δ 10.31 (s,1H), 9.99 (s, 1H), 7.59 (d, 2H), 7.56 (d, 1H), 7.37 (m, 4H), 7.29 (t,2H), 7.02 (t, 1H), 5.02 (m, 2H), 4.11 (dt, 1H), 1.90 (t, 2H), 1.61 (m,2H), 1.47 (m, 2H), 1.30 (m, 4H). MS (ESI+) calcd for C₂₂H₂₇N₃O₅ 413,found 414 [M+H]⁺.

(7S)-7-Benzyloxycarbonylamino-7-(quinolin-8-ylcarbamoyl)-heptanoic acidt-butyl ester (54)

The title compound was made from N-Cbz-L-2-aminosuberic acid 8-t-butylester, dicyclohexylamine salt in a manner similar to that for 51. Flashchromatography (0-1% MeOH/CH₂Cl₂) gave 54 as a light brown solid (70 mg,0.138 mmol, 82%). TLC R_(f) 0.42 (2% MeOH/CH₂Cl₂); ¹H-NMR (400 MHz,CDCl₃) δ 10.19 (s, 1H), 8.77 (dd, 1H), 8.71 (dd, 1H), 8.15 (dd, 1H),7.52 (m, 2H), 7.45 (m, 1H), 7.33 (m, 4H), 5.50 (br d, 1H), 5.15 (m, 2H),4.51 (br dd, 1H), 2.17 (t, 2H), 2.00 (m, 1H), 1.79 (m, 1H), 1.56 (m,2H), 1.45 (m, 2H), 1.40 (s, 9H), 1.38 (m, 2H).

(7S)-7-Benzyloxycarbonylamino-7-(quinolin-8-ylcarbamoyl)-heptanoic acid(55)

Prepared from 54 in a manner similar to that for 52. Obtained 55 as abrown solid (72 mg, 0.129 mmol). TLC R_(f) 0.16 (50% EtOAc/hexanes);¹H-NMR (400 MHz, DMSO-d₆) δ 11.92 (br s, 1H), 10.46 (s, 1H), 8.49 (dd,1H), 8.63 (dd, 1H), 8.42 (dd, 1H), 8.10 (d, 1H), 7.68 (dd, 1H), 7.58 (t,1H), 7.36 (m, 2H), 7.28 (m, 2H), 5.09 (m, 2H), 4.22 (m, 1H), 2.19 (t,2H), 1.83 (m, 1H), 1.67 (m, 1H), 1.48 (m, 2H), 1.39 (m, 2H), 1.28 (m,2H).

(1S)-[6-Hydroxycarbamoyl-1-(quinolin-8-ylcarbamoyl)-hexyl]-carbamic acidbenzyl ester (56)

Prepared from 55 in a manner similar to that for 53. Obtained 56 as awhite solid (15 mg, 0.032 mmol, 44%). ¹H-NMR (400 MHz, DMSO-d₆) δ 10.46(s, 1H), 10.31 (s, 1H), 8.85 (dd, 1H), 8.63 (dd, 1H), 8.42 (dd, 1H),8.12 (d, 1H), 8.66 (m, 2H), 7.58 (t, 1H), 7.37 (m, 2H), 7.28 (m, 2H),7.20-6.90 (1H), 5.10 (m, 2H), 4.10 (m, 1H), 1.92 (t, 2H), 1.82 (m, 1H),1.68 (m, 1H), 1.49 (m, 2H), 1.40 (m, 2H), 1.26 (m, 2H). MS (ESI+) calcdfor C₂₅H₂₈N₄O₅ 464, found 465 [M+H]⁺.

(7S)-(Cyclohexanecarbonyl-amino)-7-phenylcarbamoyl-heptanoic acid methylester (57)

To a solution of 5 (81 mg, 0.214 mmol) in CH₂Cl₂ (10 mL) was added TFA(0.5 mL) and the solution stirred for 2 h. The mixture was concentratedunder reduced pressure. To a solution of this amine (62 mg, 0.223 mmol)and cyclohexane carboxylic acid (3 μL, 0.245 mmol) in CH₂Cl₂ (4 mL) wereadded Py.BOP (140 mg, 0.268 mmol) and DIEA (58 μL, 0.335 mmol). Thesolution was stirred for 2 h, concentrated under reduced pressure, andthe product purified by flash chromatography (40% EtOAc/hexanes).Evaporation left crude 57 as a white solid (95 mg) containing a smallamount of unreacted cyclohexane acid impurity. This material was used inthe next step without further purification. TLC R_(f) 0.58 (50%EtOAc/hexanes); ¹H-NMR (400 MHz, CDCl) δ 8.58 (s, 1H), 7.50 (d, 2H),7.28 (t, 2H), 7.07 (t, 1H), 6.14 (d, 1H), 4.56 (dt, 1H), 3.64 (s, 3H),2.28 (t, 2H), 2.13 (tt, 1H), 1.94 (m, 1H), 1.85 (m, 2H), 1.76 (m, 2H),1.64 (m, 4H), 1.41 (m, 5H), 1.22 (m, 4H).

(7S)-(Cyclohexanecarbonyl-amino)-7-phenylcarbamoyl-heptanoic acid (58)

To a solution of ester 57 (95 mg) in MeOH (2.5 mL) at 0° C. was added asolution of NaOH (1 M, 2.5 mL). A white precipitate formed uponaddition, which was re-dissolved by the addition of THF (2.5 mL).Additional NaOH (1 M, 1.0 mL) was added after 3 h and the temperaturemaintained at 0° C. Upon complete disappearance of starting material byTLC analysis, the reaction contents were acidified with HCl (1 N) toobtain a white precipitate. The supernatant was drawn off, and the solidfiltered under aspiration. The combined liquors were extracted withEtOAc (3×5 mL), and the extracts combined, washed with brine, dried overMgSO₄, and filtered. Concentration under reduced pressure left a whitesolid which was combined with the filter cake and dried under vacuum toobtain the carboxylic acid 58 (75 mg, 0.200 mmol, 90%). ¹H-NMR (400 MHz,DMSO-d₆) δ 11.95 (s, 1H), 9.98 (s, 1H), 7.90 (d, 1H), 7.58 (d, 1H), 7.28(t, 2H), 7.02 (t, 1H), 4.33 (dt, 1H), 2.22 (tt, 1H), 2.17 (t, 2H), 1.67(m, 6H), 1.60 (m, 2H), 1.46 (m, 2H), 1.22 (m, 9H).

(2S)-2-(Cyclohexanecarbonyl-amino)-octanedioic acid 8-hydroxyamide1-phenylamide (59)

Acid 58 (70 mg, 0.187 mmol), TBDPSO-NH₂ (61 mg, 0.224 mmol), and DMAP (5mg) were dissolved in CH₂Cl₂ (4 mL) and EDC (47 mg, 0.243 mmol) wasadded. The solution was stirred overnight. After concentration underreduced pressure, the material was purified by flash chromatography (50%EtOAc/hexanes). Evaporation of the combined product fractions gave awhite foam (80 mg, 0.131 mmol, 70%). To a solution of this protectedhydroxamate in CH₂Cl₂ (2 mL) and THF (3 mL) was added TFA (0.25 mL) andstirred for 1.5 h. A new spot which stained immediately with FeCl₃ wasobserved on TLC. The solution was concentrated and all volatiles removedunder vacuum. The residue was triturated with EtOAc and obtain a whitegel precipitate which was transferred to a plastic tube with EtOAc (5mL). The tube was centrifuged to form a pellet, the supernatant drained,and EtOAc (10 mL) added. The pellet was resuspended with sonication,then centrifuged again, the supernatant discarded, and the residue driedunder vacuum. A white solid 59 (18 mg, 0.046 mmol, 35%) was obtained.¹H-NMR (400 MHz, DMSO-d₆) δ 10.31 (s, 1H), 9.97 (s, 1H), 7.89 (d, 1H),7.57 (d, 2H), 7.28 (t, 2H), 7.02 (t, 1H), 4.33 (dt, 1H), 2.22 (t, 2H),1.91 (t, 2H), 1.61 (m, 6H), 1.68 (m, 2H), 1.45 (m, 2H), 1.21 (9H).

Octanedioic acid hydroxyamide quinolin-8-ylamide (60)

This compound was prepared from suberic acid monomethyl ester in similarfashion to 48, with the use of 8-aminoquinoline. The crude residueobtained after TFA deprotection of the protected hydroxamate was takenup in a small volume of EtOAc and precipitated with hexanes to give 60as a white solid (18 mg, 0.057 mmol, 21% from the carboxylic acid).¹H-NMR (400 MHz, DMSO-d₆) δ 10.31 (s, 1H), 10.02 (s, 1H), 8.92 (dd, 1H),8.61 (dd, 1H), 8.40 (dd, 1H), 7.65 (dd, 1H), 7.63 (dd, 1H), 7.56 (t,1H), 2.56 (t, 1H), 1.93 (t, 1H), 1.63 (m, 2H), 1.49 (m, 2H), 1.28 (m,4H). MS (ESI+) calcd for C₁₇H₂₁N₃O₃ 315, found 316 [M+H]⁺.

2-t-Butoxycarbonyl-octanedioic acid 1-t-butyl ester 8-ethyl ester (61)

To a stirred suspension of NaH (60% disp., 197 mg, 4.913 mmol) in THF(25 mL) at 0° C. was added di-t-butyl malonate (1.00 mL, 4.466 mmol) andthe mixture allowed to warm to ambient temperature. After 1 h, gas hadceased evolving and ethyl 6-bromohexanoate (0.88 mL, 4.913 mmol) wasadded dropwise. The reaction was brought to reflux overnight. Thereaction was carefully quenched with H₂O (10 mL) and diluted with EtOAc.After separation of the layers, the aqueous portion was extracted withEtOAc (3×10 mL). The extracts were pooled and washed with H₂O, thenbrine, dried over MgSO₄, and filtered. Concentration under reducedpressure gave a yellow oil which was passed through a plug of silica gel(10% EtOAc/hexanes). Evaporation left a light yellow syrup 61 (1.52 g,4.24 mmol, 95%). TLC R_(f) 0.44 (10% EtOAc/hexanes); ¹H-NMR (400 MHz,CDCl₃) δ 4.10 (q, 2H), 3.08 (t, 1H), 2.26 (t, 2H), 1.76 (m, 2H), 1.60(m, 2H), 1.43 (s, 18H), 1.32 (m, 4H), 1.23 (m, 3H).

2-Carboxy-octanedioic acid 8-ethyl ester (62)

To a solution of triester 61 (500 mg, 1.395 mmol) in CH₂Cl₂ (20 mL) wasadded TFA (2.0 mL) and the reaction mixture stirred overnight. Volatilecomponents were evaporated under vacuum, and the residue repeatedlydissolved in CH₂Cl₂ and evaporated to remove all traces of TFA. A solid62 (327 mg, 1.33 mmol) was obtained and used directly in the next stepwithout further purification. ¹H-NMR (400 MHz, DMSO-d₆) δ 12.62 (br s,2H), 4.03 (q, 2H), 3.16 (t, 1H), 2.25 (t, 2H), 1.67 (m, 2H), 1.49 (m,2H), 1.25 (m, 4H), 1.16 (t, 3H).

7,7-Bis-(quinolin-8-ylcarbamoyl)-heptanoic acid ethyl ester (65)

Diacid 62 (150 mg, 0.609 mmol), 8-aminoquinoline (211 mg, 1.462 mmol),and DMAP (5 mg) were dissolved in THF (6 mL). To this solution was addedEDC (350 mg, 1.827 mmol) and the reaction allowed to proceed overnight.The mixture was concentrated under reduced pressure and the productpurified by flash chromatography (40% EtOAc/hexanes). Evaporation of thecombined product fractions left 63 as a light brown solid (100 mg, 0.201mmol, 14%). ¹H-NMR (400 MHz, DMSO-d₆) δ 10.85 (s, 2H), 8.92 (dd, 2H),8.64 (dd, 2H), 8.40 (dd, 2H), 7.68 (dd, 2H), 7.62 (dd, 2H), 7.57 (t,2H), 4.35 (t, 1H), 3.98 (q, 2H), 2.24 (t, 2H), 2.00 (m, 2H), 1.51 (m,2H), 1.37 (m, 4H), 1.12 (t, 3H).

7,7-Bis-(quinolin-8-ylcarbamoyl)-heptanoic acid (64)

To a solution of ester 63 (94 mg, 0.212 mmol) in MeOH (3 mL) and THF (1ml) was added a solution of LiOH.H₂O (44 mg, 1.062 mmol) in H₂O (1 mL)and the mixture was stirred for 5 h. After acidification with HCl (1 N)to pH 7, EtOAc (10 mL) was added and the layers separated. The aqueousportion was extracted with EtOAc (3×5 mL), and the extracts combined,washed with sat. NH₄Cl (3 mL), H₂O (3 mL), then brine, dried over MgSO₄,and filtered. Concentration under reduced pressure left 64 as a whitesolid (94 mg, 0.200 mmol, 94%). TLC R_(f) 0.21 (50% EtOAc/hexanes);¹H-NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 10.85 (s, 2H), 8.93 (dd, 2H),8.65 (dd, 2H), 8.40 (dd, 2H), 7.69 (dd, 2H), 7.63 (dd, 2H), 7.58 (t,2H), 4.35 (t, 1H), 2.16 (t, 2H), 2.00 (m, 2H), 1.49 (m, 2H), 1.38 (m,4H).

2-(Quinolin-8-ylcarbamoyl)-octanedioic acid 8-hydroxyamide1-quinolin-8-ylamide (65)

Acid 64 (94 mg, 0.200 mmol), TBDPSO-NH₂ (74 mg, 0.272 mmol), and DMAP (5mg) were dissolved in CH₂Cl₂ (4 mL) and EDC (57 mg, 0.295 mmol) wasadded. The solution was stirred overnight, then concentrated underreduced pressure. Purification by flash chromatography (30-50%EtOAc/hexanes) and evaporation of the combined product fractions gave awhite foam. To a solution of this protected hydroxamate in CH₂Cl₂ (4 mL)was added TFA (0.2 mL) and the solution stirred for 4 h. TLC indicatedcomplete consumption of starting material and a new spot that stainedwith FeCl₃. The solution was concentrated under reduced pressure, andthe residue dissolved in a minumum of EtOAc. Addition of hexanes gave awhite precipitate, from which the mother liquor was removed. Afterrinsing with hexanes, the residue was dried under vacuum to leave 65 asa white solid (30 mg, 0.061 mmol, 22% from the carboxylic acid). ¹H-NMR(400 MHz, CDCl₃) δ 10.85 (s, 2H), 10.30 (s, 1H), 8.93 (dd, 2H), 8.65(dd, 2H), 8.40 (dd, 2H), 7.69 (dd, 2H), 7.63 (dd, 2H), 7.58 (t, 2H),4.35 (t, 1H), 1.99 (m, 2H), 1.92 (t, 2H), 1.48 (m, 2H), 1.35 (m, 4H). MS(ESI+) calcd for C₂₇H₂₇N₅O₄ 485, found 486 [M+H]⁺.

2-(Quinolin-3-ylcarbamoyl)-octanedioic acid 8-hydroxyamide1-quinolin-3-ylamide (68)

The title compound was made from diacid 62 as analogous to 65. ¹H-NMR(400 MHz, DMSO-d₆) δ 10.60 (s, 1H), 10.34 (s, 1H), 8.95 (dd, 2H), 8.74(s, 2H), 7.93 (dd, 2H), 7.64 (dd, 2H), 7.56 (dd, 2H), 3.71 (t, 1H), 1.96(m, 4H), 1.51 (m, 2H), 1.34 (m, 4H).

6-Bromohexanoic acid phenylamide (76)

To a solution of 6-bromohexanoyl chloride (1.00 mL, 6.53 mmol) in THF(35 mL) at 0° C. was added dropwise a solution of aniline (0.60 mL, 6.53mmol) and TEA (1.09 mL, 7.84 mmol) in THF (5 mL). The reaction mixturewas allowed to warm to ambient temperature and stirred for 2 h. Themixture was filtered, the solids rinsed with EtOAc, and the filtratereduced under vacuum. The residue was partitioned between H₂O (15 mL)and EtOAc (20 mL) and the layers separated. The aqueous portion wasextracted with EtOAc (3×10 mL) and the organic layers combined, washedwith HCl (1 N), brine, dried over MgSO₄, and filtered. Concentrationunder reduced pressure left a brown oil which was passed through a plugof silica gel (30% EtOAc/hexanes) under aspiration. Concentration underreduced pressure left 67 as a solid (1.55 g, 5.74 mmol, 88%). TLC R_(f)0.36 (25% EtOAc/hexanes); ¹H-NMR (400 MHz, DMSO-d₆) δ 9.85 (s, 1H), 7.57(d, 2H), 7.27 (t, 2H), 7.01 (t, 1H), 3.53 (t, 2H), 2.30 (t, 2H), 1.81(t, 2H), 1.63 (m, 2H), 1.42 (m, 2H); MS (ESI+) calcd for C₁₂H₁₆BrNO268+270, found 269+271 [M+H]⁺.

Thioacetic acid S-(5-phenylcarbamoyl-pentyl) ester (68)

Bromide 67 (200 mg, 0.74 mmol), potassium thioacetate (110 mg, 0.96mmol), and sodium iodide (10 mg) were combined in THF (6 mL) and thevigorously stirred mixture brought to reflux overnight. The reactionmixture was concentrated, the passed through a plug of silica gel (20%EtOAc/hexanes, 200 mL) under aspiration. Evaporation under reducedpressure left 68 as an orange crystalline solid (190 mg, 0.72 mmol,97%). TLC R_(f) 0.22 (25% EtOAc/hexanes); ¹H-NMR (400 MHz, DMSO-d₆) δ9.83 (s, 1H), 7.56 (d, 2H), 7.27 (t, 2H), 7.00 (t, 1H), 2.82 (t, 2H),2.30 (s, 3H), 2.28 (t, 2H), 1.57 (m, 2H), 1.52 (m, 2H), 1.35 (m, 2H).

6-Methanesulfonylamino-hexanoic acid (69)

6-aminohexanoic acid (904 mg, 6.89 mmol) and NaOH (415 mg, 10.34 mmol)were dissolved in H₂O (30 mL) and cooled to 0-5° C. Methanesulfonylchloride (0.586 mL, 7.58 mmol) was added dropwise and the reactionmixture stirred for 2 h, then warmed to ambient temperature and stirredfor an additional 2 h. The mixture was acidified with HCl (1 N) andextracted with EtOAc (3×15 mL). The extracts were combined, washed withH₂O, then brine, dried over MgSO₄, and filtered. Evaporation underreduced pressure gave 69 as a white crystalline solid (207 mg, 0.99mmol, 14%). ¹H-NMR (400 MHz, DMSO-d₆) δ 11.95 (s, 1H), 6.91 (t, 1H),2.90 (dt, 2H), 2.87 (s, 3H), 2.20 (t, 2H), 2.48 (m, 2H), 2.43 (m, 2H),1.27 (m, 2H).

6-Methanesulfonylamino-hexanoic acid phenylamide (70)

To a solution of acid 69 (100 mg, 0.48 mmol), aniline (60 μL, 0.66mmol), and DMAP (5 mg) in THF (5 mL) was added EDC (119 mg, 0.57 mmol).The reaction mixture was stirred overnight, then partitioned between H₂O(10 mL) and EtOAc (15 mL). The layers were separated, and the aqueousportion extracted with EtOAc (3×10 mL). The organic fractions werecombined, washed with sat. NH₄Cl (5 mL), then brine, dried over MgSO₄,and filtered. Concentration under reduced pressure gave 70 as a whitecrystalline solid (130 mg, 0.46 mmol, 95%). ¹H-NMR (400 MHz, DMSO-d₆) δ9.84 (s, 1H), 7.57 (d, 2H), 7.26 (t, 2H), 7.00 (t, 1H), 6.92 (t, 1H),2.91 (dt, 2H), 2.85 (s, 3H), 1.58 (m, 2H), 1.47 (m, 2H), 1.31 (m, 2H).

9,9,9-trifluoro-8-oxononanoic acid methyl ester (71)

To a solution of suberic acid monomethyl ester (1.00 g, 5.31 mmol) inTHF (15 mL) was added oxalyl chloride (2 mL) followed by DMF (1 drop).The solution was stirred for 2 h, then concentrated under reducedpressure. Volatiles were removed under high vacuum overnight, leaving ayellow oil (1.08 g, 5.22 mmol, 98%). This crude acid chloride was thentransformed into the trifluoromethyl ketone by a literature method asfollows. (65) To a solution of the acid chloride (1.08 g, 5.22 mmol) inCH₂Cl₂ (45 mL) at 0° C. were added trifluoroacetic anhydride (4.64 mL,32.81 mmol) and pyridine (3.54 mL, 43.74 mmol). The mixture was allowedto warm to ambient temperature and stirred for 2 h. After returning to0° C., ice-cold H₂O (20 mL) was added carefully. Additional H₂O (100 mL)was added and the layers separated. The aqueous phase was extracted withCH₂Cl₂ (2×30 mL) and the organic layers combined, washed with brine,dried over MgSO₄, and filtered. Evaporation under reduced pressure lefta brown oil, which was purified by flash chromatography (2-4%MeOH/CH₂Cl₂) to give 71 as a clear oil (641 mg, 2.67 mmol, 49%). TLCR_(f) 0.24 (2% MeOH/CH₂Cl₂); ¹-NMR (400 MHz, CDCl₃) δ 3.67 (s, 3H), 2.71(t, 2H), 2.31 (t, 2H), 1.65 (m, 4H), 1.35 (m, 4H).

9,9,9-Trifluoro-8-oxo-nonanoic acid phenylamide (72)

To a solution of ester 71 (300 mg, 1.25 mmol) in THF (18 mL) was added asolution of LiOH.H₂O (262 mg, 6.24 mmol) in H₂O (6 mL) and thesuspension was stirred overnight. The mixture was then acidified withHCl (1 N) to pH 2 and then extracted with EtOAc (3×15 mL). The extractswere combined, washed with brine, dried over MgSO₄, and filtered.Concentration under reduced pressure left a white solid (211 mg, 0.93mmol, 75%). To a solution of this acid (109 mg, 0.48 mmol), EDC (111 mg,0.58 mmol), and DMAP (5 mg) in CH₂Cl₂ (5 mL) was added aniline (49 μL,0.53 mmol) and the reaction allowed to proceed overnight. The solutionwas partitioned between H₂O (5 mL) and EtOAc (10 mL). The layers wereseparated, and the aqueous phase extracted with EtOAc (3×5 mL). Theorganic portions were combined, washed with brine, dried over MgSO₄, andfiltered. Evaporation under reduced pressure left a solid which waspurified by preparative TLC (30% EtOAC/hexanes) with isolation of theleast polar band by EtOAc extraction. The extract was concentrated togive 72 as a yellowish solid (92 mg, 0.31 mmol, 65%). TLC R_(f) 0.48(50% EtOAc/hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.51 (d, 2H), 7.32 (t,2H), 7.10 (t, 1H), 2.72 (t, 2H), 2.36 (t, 2H), 1.72 (m, 4H), 1.40 (m,4H); ¹⁹F NMR (? MHz, CDCl₃) −78.40 (s, 3F); MS (APCI+) calcd forC₁₅H₁₉F₃NO₂ 301, found 325 [M+Na]⁺.

(5-Phenylcarbamoyl-pentyl)-carbamic acid t-butyl ester (73)

To a solution of N-Boc-6-aminohexanoic acid (2.50 g, 10.81 mmol), EDC(2.69 g, 14.05 mmol), and DMAP (20 mg) in CH₂Cl₂ (100 mL) was addedaniline (1.04 mL, 11.35 mmol) and the mixture stirred overnight. Thesolution was evaporated under reduced pressure to a small volume, thenpartitioned between H₂O (20 mL) and EtOAc (30 mL). The layers wereseparated, and the aqueous phase extracted with EtOAc (3×15 mL). Theorganic portions were combined, washed with sat. NH₄Cl (5 mL), thenbrine, dried over MgSO₄, and filtered. Concentration under reducedpressure left pure 73 as a white solid (3.14 g, 10.25 mmol, 95%). TLCR_(f) 0.40 (50% EtOAc/hexanes); ¹H-NMR (400 MHz, DMSO-d₆) δ 9.81 (s,1H), 7.56 (d, 2H), 7.26 (t, 2H), 7.00 (t, 1H), 6.74 (t, 1H), 2.89 (dt,2H), 2.27 (t, 2H), 1.56 (m, 2H), 1.38 (m, 2H), 1.35 (s, 9H), 1.25 (m,2H).

6-Aminohexanoic acid phenylamide, TFA salt (74)

To a solution of carbamate 73 (300 mg, 0.98 mmol) in CH₂Cl₂ (15 mL) wasadded TFA (0.75 mL) and the solution stirred overnight. Completeconsumption of starting material was confirmed by TLC. The mixture wasevaporated under reduced pressure to remove all volatiles, leaving anoff-white solid (295 mg, 0.92 mmol, 94%). Crude 74 was used withoutfurther purification.

N-(N-Phenylcarbamoyl-5-pentyl)phosphoramidic acid dimethyl ester (75)

To a stirred suspension of ammonium salt 74 (197 mg, 0.62 mmol) and DIEA(148 μL, 0.85 mmol) in CH₂Cl₂ (7 mL) at 0° C. was added dropwisedimethyl chlorophosphate (77 μL, 0.72 mmol). The mixture was allowed towarm to ambient temperature and stirred overnight. The solution wasdiluted with H₂O (10 mL) and the layers separated. The aqueous phase wasextracted with CH₂Cl₂ (3×10 mL), the organic portions combined, washedwith sat. NH₄Cl (5 mL), then brine, dried over MgSO₄, and filtered.After concentration, the residue was purified by flash chromatography(2-5% MeOH/CH₂Cl₂), and the fractions containing the more polar of thetwo UV-active bands on TLC were combined and concentrated, giving 75 asa clear oil (40 mg, 0.13 mmol, 20%). TLC R_(f) 0.23 (5% MeOH/CH₂Cl₂);¹H-NMR (400 MHz, DMSO-d₆) δ 9.84 (s, 1H), 7.57 (d, 2H), 7.26 (t, 2H),7.00 (t, 1H), 4.90 (dt, 1H), 3.51 (d, 6H), 2.71 (m, 2H), 2.28 (t, 2H),1.56 (m, 2H), 1.40 (m, 2H), 1.29 (m, 2H).

Methyl N-(5-N-phenylcarbamoylpentyl)methylphosphonamidate (76)

To a suspension of ammonium salt 74 (155 mg, 0.48 mmol) in CH₃CN (8 mL)were added DIEA (0.21 mL) and methyl methylphosphonochloridate (77 mg,0.600 mmol). The reaction mixture was stirred overnight, during whichtime it clarified. The solution was partitioned between H₂O (10 mL) andEtOAc (15 mL) and the layers separated. The aqueous portion wasextracted with EtOAc (3×10 mL) and the organics combined, washed withsat. NH₄Cl (1×5 mL), then brine, dried over MgSO, and filtered. Theproduct was purified by flash chromatography (3-10% MeOH/CH₂Cl₂), andthe fractions containing the more polar spot were combined andconcentrated to give 76 as a clear oil (102 mg, 0.34 mmol, 71%). TLCR_(f) 0.16 (5% MeOH/CH₂Cl₂); ¹H-NMR (400 MHz, DMSO-d₆) δ 9.85 (s, 1H),7.57 (d, 2H), 7.26 (t, 2H), 7.00 (t, 1H), 4.52 (dt, 1H), 3.43 (d, 3H),2.73 (m, 2H), 2.28 (t, 2H), 1.57 (m, 2H), 1.38 (m, 2H), 1.28 (m, 2H),1.26 (d, 3H).

Example 18 Synthesis of Compound 77 Diethyl 3-bromophenylmalonate

Diethyl 3-bromophenyl malonate was prepared according to the proceduresof Cehnevert, R. and Desjardins, M. Can. J. Chem. 1994. 72, 3212-2317.¹H NMR (CDC13, 300 MHz)δ 7.6 (s, 1H), 7.50 (d, 1H, J-7.9 Hz), 7.37 (d,1H, J=7.9 Hz), 7.26 (t, 1H, J=7.9 Hz), 4.58 (s, 1H), 4.22 (m, 4H), 1.29(t, J=10 Hz).

3-bromophenyl malonyl di(phenylamide)

Diethyl 3-bromophenyl malonate (1 g, 3.2 mmol) was added to aniline (5mL). The reaction mixture was purged with Ar (g) and brought to refluxfor 2 h. After cooling, the reaction mixture was diluted with 10% HCl(20 mL) and ethyl acetate (50 mL). The organic layer was separated andconcentrated to afford 3-bromophenyl malonyl di(phenylamide) as a whitepowder. (540 mg. 1.3 mmol, 42%). ¹H NMR (d6-DMSO, 300 MHz) δ 10.3 (bs,2H), 7.65)s, 1H), 7.60 (d, 4H, J=7.9 Hz), 7.54 (d, 1H, J=7.9 Hz), 7.46(d, 1H, J=7.8 Hz), 7.35 (t, 1H, J=7.8 Hz), 7.31 (t, 4H, J=7.8 Hz), 7.06(t, 2H, J=7.6 Hz), 4.91 9s, 1H).

3-(malonyl di(phenylamide)) cinnamic acid

3-bromophenyl malonyl di(phenylamide) (500 mg, 1.22 mmol), acrylic acid(115 mg, 1.6 mmol, 1.3 equiv.), Pd(OAc)₂ (2 mg), tri-o-tolyl phosphone(20 mg), tributyl amine (0.6 mL) and xylenes (5 mL) were heated to 120°C. for 6 h in a sealed vessel. After cooling, the reaction was dilutedwith 5% HCl (10 mL) and ethyl acetate (50 mL). The organic layer wasseparated, filtered and on standing 3-(malonyl di(phenylamide))cinnamicacid precipitated as a white powder (450 mg, 1.12 mmol, 92%). ¹H NMR(d6-DMSO, 300 MHz, δ 12.4 (bs, 1H), 10.3 (bs, 2H), 7.73 (s, 1H), 7.7-7.5(m, 6H), 7.52 (d, 1H, J=7.7 Hz), 7.43 (t, 1H, J=7.6 Hz), 7.31 (t, 4H,J=7.5 Hz), 7.06 (t, 2H, J=7.4 Hz), 6.52 (d, 1H, J=16 Hz), 4.95 (s, 1H).APCI-MS 401 (M+1).

3-(malonyl di(phenylamide)) cinnamyl hydroxamic acid (77)

3-(malonyl di(phenylamide)) cinnamic acid (200 mg, 0.5 mmol) wasdissolved in dry CH₂CL₂ (10 mL). Isobutylchloroformate (0.10 mL, 0.77mmol) and triethyl amine (0.20 mL) were added at 0° C. with stirring.After 2 h at 25° C., O-(t-butyldiphenyl silyl)hydroxylamine was addedand the mixture was stirred an additional 4 h. The crude reactionmixture was applied directly to a pad a silica gel (15 g) and elutionwith 20% ethyl acetate/hexanes afforded the corresponding silylprotected hydroxamic acid (Rf=0.58, 50% ethyl acteate/hexanes) as afoam. This was treated directly with 10% trifluoracetic acid indichloromethane (10 mL) for 4 h. The solvents were concentrated at 50°C. by rotavap and the residue was suspended in ethyl-ether (10 mL).Filtration of the resultant precipitate afforded compound 77 as a whitepowder (150 mg, 0.365 mmol, 73%). ¹H NMR (d6-DMSO, 300 MHz, δ 10.8 (bs,0.5H), 10.2 (bs, 2H), 9.06 (bs, 0.5H), 7.7-7.55 (m,5H), 7.53-7.38 (m,4H), 7.31 (t, 4H, J=7.7 Hz), 7.06 (t, 2H, J=7.3 Hz), 6.50 (d, 1H, J=16Hz), 4.92 (s, 1H). APCI-MS 416 (M+1).

The effect of compound 77 on MEL cell differentiation and HistoneDeacetylase activity is shown in Table 2. Compound 77 corresponds tostructure 683 in Table 2. As evident from Table 2, compound 77 isexpected to be a highly effective cytodifferentiating agent.

Results

All the compounds which were prepared were tested. Table 2 below showsthe results of testing of only a subgroup of compounds. Table 2 iscompiled from experiments similar to the experiments described inExamples 7-10 above. The tested compounds were assigned structurenumbers as shown in Table 2. The structure numbers were randomlyassigned and do not correlate to the compound numbers used elsewhere inthis disclosure.

The results shown in Table 2 verify the general accuracy of thepredictive principals for the design of compounds having celldifferentiation and HDAC inhibition activity discussed above in thisdisclosure. Based on the principals and synthesis schemes disclosed, anumber of additional compounds can readily be designed, prepared andtested for cell differentiation and HDAC inhibition activity.

FIGS. 11 a-f show the effect of selected compounds on affinity purifiedhuman epitope-tagged (Flag) HDAC1. The effect was assayed by incubatingthe enzyme preparation in the absence of substrate on ice for 20 minuteswith the indicated amounts of compound. Substrate([³H]acetyl-labeledmurine erythroleukemia cell-derived histones) was added and the sampleswere incubated for 20 minutes at 37° C. in a total volume of 30 μl. Thereactions were then stopped and released acetate was extracted and theamount of redioactivity released determined by scintillation counting.This is a modification of the HDAC Assay described in Richon et al. 1998(39).

TABLE 2 Inhibition data of selected compounds. MEL Diff % cells/ HDACinh NO: Structure Range Opt. B+ mlx 10⁻⁵ Range ID50 SAHA(390)

0.5 to 50μM 2.5 μM 68 3.6 0.001 to100 μM 200 nM 654

0.1 to 50μM 200 nM 44 9 0.0001to 100μM 1 mM 655

0.1 to 50μM 400 nM 16 3.3 0.01 to100 μM 100 nM 656

0.4 to 50μM 0 0.01 to100 μM >100 μM 657

0.4 to 50μM 0 0.01 to100 μM >100 μM 658

0.01 to50 μM 40 nM 8 13 0.0001to 100 μM 2.5 nM 659

0.4 to 50μM 0 0.01 to100 μM 10 μM 660

0.2 to12.5 μM 800 nM 27 0.001 to100 μM 50 nM 661

0.1 to50 μM 500 nM 7 0.01 to100 μM 20 nM 662

0.2 to50 μM 0 0.001 to100 μM >100 μM MEL Cell Differentiation cells/HDACl Inhibition No. Structure Range Opt. % B+ mlx 10⁻⁵ Range ID50 663

0.2 to50 μM 200 nM 43 7 0.001 to100 μM 100 nM 664

0.2 to50 μM 400 nM 33 22 0.001 to100 μM 50 nM 665

0.1-50μM 150 nM 24 30 0.001 to100 μM 50 nM 666

0.1-50μM 150 nM 31 28 0.001 to100 μM 100 nM 667

0.02-10 μM 80 nM 27 2 0.001 to100 μM 50 nM 668

0.02 to10 μM 10 μM 11 4.7 0.001 to100 μM 100 nM 669

0.8 to50 μM 4 μM 11 16.0 0.001 to100 μM 10 μM 670

0.4 to 50μM Noeffectup to25 μM — 13.0 0.001 to100 μM >100 μM 671

0.4 to 50μM 3.1 μM 35 12.5 0.001 to100 μM 200 nM 672

0.8 to 50μM 0 No Inh 0.01 to100 μM 100 μM 673

0.8 to 50μM 0 No Inh 0.01 to100 μM 100 μM 674

0.8 to 50μM 0 Dead at25 μM 0.01 to100 μM 50 μM 675

0.8 to 50μM 0 No Inh 0.001 to100 μM >100 μM 676

0.8 to 50μM 0 No Inh 0.01 to100 μM 100 μM 677

0.05 to25 μM 1.6 μM 23 4.5 0.001 to100 μM 5 nM MEL cell differentiationcells/ml HDAC Inh No. Structure Range Opt. % B+ x 10 − 5 Range ID50 678

0.8 to 50μM 0 No Inh 0.001 to100 μM >100 μM 679

0.8 to 50μM 0 No inh 0.001 to100 μM >100 μM 680

0.01 to100 μM >100 μM 681

0.8 to 50μM 3 μM 3 2.5 0.01 to100 μM 200 nM 682

0.8 to 50μM 50 μM 8 1.1 0.01 to100 μM 150 nM 683

0.01 to0.1 μM 20 nM 9 9.0 0.0001to 100μM 1 nM 684

0.4 to 50μM 0 No inh 0.01 to100 μM 100 μM 685

0.125 to5 μM 1.0 μM 20 1.0 0.01 to100 μM 150 nM 686

0.4 to 50μM 0 No inh 0.01 to100 μM 100 μM 687

0.125 to5 μM 0 No inh 0.01 to100 μM 200 nM 688

0.4 to 50μM 0 No inh 0.01 to100 μM >100 μM 689

5.0 to 40μM 35 μM 48 2.0 0.01 to100 μM 200 nM 690

5.0 to 40μM 10 μM 38 2.5 0.01 to100 μM 150 nM 691

10 to 25μM 0 No inh 0.01 to100 μM 100 nM 692

0.03 to 5μM 1 μM 27 18.0  0.01 to100 μM 1 nM 693

0.4 to 50μM 0 No inh 0.01 to100 μM >100 μM

BIBLIOGRAPHY

-   1. Sporn, M. B., Roberts, A. B., and Driscoll, J. S. (1985) in    Cancer: Principles and Practice of Oncology, eds. Hellman, S.,    Rosenberg, S. A., and DeVita, V. T., Jr., Ed. 2, (J.B. Lippincott,    Philadelphia), P. 49.-   2. Breitman, T. R., Selonick, S. E., and Collins, S. J. (1980) Proc.    Natl. Acad. Sci. USA 77: 2936-2940.-   3. Olsson, I. L. and Breitman, T. R. (1982) Cancer Res. 42:    3924-3927.-   4. Schwartz, E. L. and Sartorelli, A. C. (1982) Cancer Res. 42:    2651-2655.-   5. Marks, P. A., Sheffery, M., and Rifkind, R. A. (1987) Cancer Res.    47: 659.-   6. Sachs, L. (1978) Nature (Lond.) 274: 535.-   7. Friend, C., Scher, W., Holland, J. W., and Sato, T. (1971) Proc.    Natl. Acad. Sci. (USA) 68: 378-382.-   8. Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R. A.,    and Marks, P. A. (1975) Proc. Natl. Acad. Sci (USA) 72: 1003-1006.-   9. Reuben, R. C., Wife, R. L., Breslow, R., Rifkind, R. A., and    Marks, P. A. (1976) Proc. Natl. Acad. Sci. (USA) 73: 862-866.-   10. Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K.,    Yamazaki, T., Yoshika, S., and Suda, T. (1981) Proc. Natl, Acad,    Sci. (USA) 78: 4990-4994.-   11. Schwartz, E. L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and    Sartorelli, A. C. (1983) Proc. Am. Assoc. Cancer Res. 24: 18.-   12. Tanenaga, K., Hozumi, M., and Sakagami, Y. (1980) Cancer Res.    40: 914-919.-   13. Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15: 731-740.-   14. Metcalf, D. (1985) Science, 229: 16-22.-   15. Scher, W., Scher, B. M., and Waxman, S. (1983) Exp. Hematol. 11:    490-498.-   16. Scher, W., Scher, B. M., and Waxman, S. (1982) Biochem. &    Biophys. Res. Comm. 109: 348-354.-   17. Huberman, E. and Callaham, M. F. (1979) Proc. Natl. Acad. Sci.    (USA) 76: 1293-1297.-   18. Lottem, J. and Sachs, L. (1979) Proc. Natl. Acad. Sci. (USA) 76:    5158-5162.-   19. Terada, M., Epner, E., Nudel, U., Salmon, J., Fibach, E.,    Rifkind, R. A., and Marks, P. A. (1978) Proc. Natl. Acad. Sci. (USA)    75: 2795-2799.-   20. Morin, M. J. and Sartorelli, A. C. (1984) Cancer Res. 44:    2807-2812.-   21. Schwartz, E. L., Brown, B. J., Nierenberg, M., Marsh, J. C., and    Sartorelli, A. C. (1983) Cancer Res. 43: 2725-2730.-   22. Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y. (1973)    Bibl. Hematol. 39: 943-954.-   23. Ebert, P. S., Wars, I., and Buell, D. N. (1976) Cancer Res. 36:    1809-1813.-   24. Hayashi, M., Okabe, J., and Hozumi, M. (1979) Gann 70: 235-238.-   25. Fibach, E., Reuben, R. C., Rifkind, R. A., and    Marks, P. A. (1977) Cancer Res. 37: 440-444.-   26. Melloni, E., Pontremoli, S., Damiani, G., Viotti, P., Weich, N.,    Rifkind, R. A., and Marks, P. A. (1988) Proc. Natl. Acad. Sci. (USA)    85: 3835-3839.-   27. Reuben, R., Khanna, P. L., Gazitt, Y., Breslow, R., Rifkind, R.    A., and Marks, P. A. (1978) J. Biol. Chem. 253: 4214-4218.-   28. Marks, P. A. and Rifkind, R. A. (1988) International Journal of    Cell Cloning 6: 230-240.-   29. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O.,    Cakiroglu, A. G., Jackson, J. F., Rifkind, R. A., and    Marks, P. A. (1987) Proc. Natl. Acad. Sciences (USA) 84: 5282-5286.-   30. Marks, P. A. and Rifkind, R. A. (1984) Cancer 54: 2766-2769.-   31. Egorin, M. J., Sigman, L. M. VanEcho, D. A., Forrest, A.,    Whitacre, M. Y., and Aisner, J. (1987.) Cancer. Res. 47: 617-623.-   32. Rowinsky, E. W., Ettinger, D. S., Grochow, L. B., Brundrett, R.    B., Cates, A. E., and Donehower, R. C. (1986) J. Clin. Oncol. 4:    1835-1844.-   33. Rowinsky, E. L. Ettinger, D. S., McGuire, W. P., Noe, D. A.,    Grochow, L. B., and Donehower, R. C. (1987) Cancer Res. 47:    5788-5795.-   34. Callery, P. S., Egorin, M. J., Geelhaar, L. A., and    Nayer, M. S. B. (1986) Cancer Res. 46: 4900-4903.-   35. Young, C. W. Fanucchi, M. P., Walsh, T. B., Blatzer, L.,    Yaldaie, S., Stevens, Y. W., Gordon, C., Tong, W., Rifkind, R. A.,    and Marks, P. A. (1988) Cancer Res. 48: 7304-7309.-   36. Andreeff, M., Young, C., Clarkson, B., Fetten, J., Rifkind, R.    A., and Marks, P. A. (1988) Blood 72: 186a.-   37. Marks, P. A., Richon, V. M., Breslow, R., Rifkind, R. A., Life    Sciences 1999, 322: 161-165.-   38. Yoshida et al., 1990, J. Biol. Chem. 265:17174-17179.-   39. Richon, V. M., Emiliani, S., Verdin, E., Webb, Y., Breslow, R.,    Rifkind, R. A., and Marks, P. A., Proc. Natl. Acad. Sci. (USA) 95:    3003-3007 (1998).-   40. Nishino, N. et. al. Chem. Pharm. Bull. 1996, 44, 212-214.-   41. U.S. Pat. No. 5,369,108, issued Nov. 29, 1994.-   42. Kijima et al., 1993, J. Biol. Chem. 268:22429-22435.-   43. Lea et al., 1999, Int. J. Oncol. 2:347-352.-   44. Kim et al., 1999, Oncogene 15:2461-2470.-   45. Saito et al., 1999, Proc. Natl. Acad. Sci. 96:4592-4597.-   46. Lea and Tulsyan, 1995, Anticancer Res. 15:879-883.-   47. Nokajima et al., 1998, Exp. Cell Res. 241:126-133.-   48. Kwon et al., 1998, Proc. Natl. Acad. Sci. USA 95:3356-3361.-   49. Richon et al, 1996, Proc. Natl. Acad. Sci. USA 93:5705-5708.-   50. Kim et al., 1999, Oncogene 18:2461-2470.-   51. Yoshida et al., 1995, Bioessays 17:423-430.-   52. Yoshida & Beppu, 1988, Exp. Cell. Res. 177:122-131.-   53. Warrell et al., 1998, J. Natl. Cancer Inst. 90:1621-1625.-   54. Desai et al., 1999, Proc. AACR 40: abstract #2396.-   55. Cohen et al., Antitumor Res., submitted.-   56. D. W. Christianson and W. N. Lipscomb, “The Complex Between    Carboxypeptidase A and a Possible Transition-State Analogue:    Mechanistic Inferences from High-Resolution X-ray Structures of    Enzyme-inhibitor Complexes,” J. Am. Chem. Soc. 1986, 108, 4998-5003.-   57. G. H. S. Prakash and A. K. Yudin, “Perfluoroalkylation with    organosilicon Reagents,” Chem. Rev. 1997, 97, 757-786.-   58. J.-C. Blazejewski, E. Anselmi, and M. P. Wilmshurst, “Extending    the Scope of Ruppert's Reagent: Trifluoromethylation of Imines,”    Tet. Letters 1999, 40, 5475-5478.-   59. R. J. Linderman and D. M. Graves, “Oxidation of    Fluoroalkyl-Substituted Carbinols by the Dess-Martin Reagent,” J.    Org. Chem. 1989, 54, 661-668.-   60. N. E. Jacobsen and P. A. Bartlett, “A Phosphonamidate Dipeptide    Analogue as an Inhibitor of Carboxypeptidase A,” J. Am. Chem. Soc.    1981, 103, 654-657.-   61. S. Lindskog, L. E. Henderson, K. K. Kannan, A. Liljas, P. O.    Nyman, and B. Strandberg, “Carbonic Anhydrase”, in The Enzymes, 3rd    edition, P. D. Boyer, ed., 1971, vol. V, pp. 587-665, see p. 657.-   62. Durrant, G.; Greene, R. H.; Lambeth, P. F.; Lester, M. G.;    Taylor, N. R., J. Chem. Soc., Perkin Trans. I 1983, 2211-2214.-   63. Burden, R. S.; Crombie, L., J. Chem. Soc. (C) 1969, 2477-2481.-   64. Farquhar, D.; Cherif, A.; Bakina, E.; Nelson, J. A., J. Med.    Chem., 1998, 41, 965-972.-   65. Boivin, J.; El Kaim, L.; Zard, S. Z., Tet. Lett. 1992, 33,    1285-1288.-   66. Finnin, M. S. et al., Structures of a histone deacetylase    homologue bound to the TSA and SAHA inhibitors. Nature 401, 188-93    (1999).-   67. Webb, Y. et al., Photoaffinity labeling and mass spectrometry    identify ribosomal protein S3 as a potential target for hybrid polar    cytodifferentiation agents. J. Biol. Chem. 274, 14280-14287 (1997).-   68. Butler, L. M. et al., Suberoylanilide hydroxamic acid (SAHA), an    inhibitor of histone deacetylase, suppresses the growth of the CWR22    human prostate cancer xenograft. submitted (2000).

1. A compound having the formula:

wherein R₁ and R₂ are the same or different and are each attachedthrough a linker, and are substituted or unsubstituted aryl, substitutedor unsubstituted cycloalkyl, cycloalkylamino, naphthyl, pyridineamino,piperidino, 9-purine-6-amino, thiazoleamino, hydroxyl, branched orunbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or pyridinylgroup; wherein R₅′ is —C(O)—CF₃ (trifluoroacetyl); wherein the linker isan amide moiety, —O—, —S—, —NH—, or —CH₂—; and n is an integer from 3 to10, or an enantiomer or a pharmaceutically acceptable salt thereof.
 2. Acompound having the formula:

wherein each of R₇ is substituted or unsubstituted aryl, substituted orunsubstituted cycloalkyl, cycloalkylamino, naphthyl, pyridineamino,piperidino, 9-purine-6-amino, thiazoleamino, hydroxyl, branched orunbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or pyridinylgroup or wherein —NH—R₇ is replaced by a moiety selected from the groupconsisting of:

wherein R₂ is -sulfonamide-R₈, or -amide-R₈, wherein R₈ is substitutedor unsubstituted aryl, substituted or unsubstituted cycloalkyl,cycloalkylamino, naphthyl, pyridineamino, piperidino, 9-purine-6-amino,thiazoleamino, hydroxyl, branched or unbranched alkyl, alkenyl,alkyloxy, aryloxy, arylalkyloxy, or pyridinyl group; and wherein R₅ is—C(O)—CF₃ (trifluoroacetyl); and n is an integer from 3 to 10, or anenantiomer or a pharmaceutically acceptable salt thereof.
 3. Thecompound of claim 2, wherein R₂ is —NH—C(O)—Y or —NH—SO₂—Y, and whereinY is selected from the group consisting of:


4. The compound of claim 2, wherein R₇ is selected from the groupconsisting of:


5. A pharmaceutical composition comprising the compound of any one ofclaims 1-4 and a pharmaceutically acceptable carrier.
 6. Apharmaceutically acceptable salt of the compound of claim
 2. 7. Thecompound of claim 2, wherein —NH—R₇ is selected replaced by a moietyfrom the group consisting of:


8. The compound of claim 2, wherein R₇ is a phenyl and R₂ is -amide-R8,wherein R₈ is quinoline.
 9. The compound of claim 1 having the formula:

or a pharmaceutically acceptable salt or enantiomer thereof.